Acute lymphoblastic leukemia (ALL) is a common cancer affecting children worldwide. The development of ALL is driven by several genes, some of which can be targeted for treatment by inhibiting gene fusions. PAX5 is frequently mutated in ALL and is involved in chromosomal rearrangements and translocations. Mutations in PAX5 interact with other genes, such as ETV6 and FOXP1, which influence B-cell development. PAX5/ETV6 has been observed in both B-ALL patients and a mouse model. The interaction between PAX5 and FOXP1 negatively suppresses the Pax5 gene in B-ALL patients.Additionally, ELN and PML genes have been found to fuse with PAX5, leading to adverse effects on B-cell differentiation. ELN-PAX5 interaction results in the decreased expression of LEF1, MB1, and BLNK, while PML-PAX5 is critical in the early stages of leukemia. PAX5 fusion genes prevent the transcription of the PAX5 gene, making it an essential target gene for the study of leukemia progression and the diagnosis of B-ALL. Research suggests that the highest likelihood of ALL and its pre-B subtype is in children under 5 years old, with the risk declining until the mid-twenties, which starts to increase again. At the mechanistic level, mutations that arise during the differentiation of blood cells are responsible for the onset of ALL. Consequently, these DNA mutations in white blood cells lead to their uncontrollable growth and proliferation. This change can be passed on genetically from parent to child, or it can arise accidentally due to exposure to hazardous substances.,6Acute lymphoblastic leukemia (ALL) is a malignant disease that develops from the lymphoid precursor cells in the bone marrow. Over a third (34.1%) of all childhood cancer disability-adjusted life years are attributed to ALL, making it the most common childhood cancer worldwide. Swyer syndrome, and Bainbridge-Ropers syndrome. It has been indicated earlier that there is a correlation between numerous germline alleles, variants passed down from parent to child, and somatic mutations in children with ALL.,11 Several alleles have been linked to ALL, including IKZF1, PAX5, and ETV6 in the germline, and KRAS, NRAS, PTPN11, JAK2, and FLT3,16 in the somatic mutations.Moreover, several inherited disorders have been linked to ALL, including Down syndrome, ALL have 2 primary subtypes, B- and T-ALL, which have recently been further classified based on whole transcriptome analysis and gene expression grouping using contemporary clinical, cytogenetic, and molecular data. Among ALL subtypes, those originating from the B-cell lineage are the most prevalent, accounting for approximately 85% of cases. However, new B-ALL and T-ALL subtypes have recently been identified, some of which are defined by unique gene rearrangements or a single gene mutation. These novel subtypes have demonstrated distinct clinical features and responses to treatment.Despite remarkable progress in treating ALL over the past decades, the disease remains a significant challenge in oncology. This challenge is due to its heterogeneity, with diverse clinical and biological characteristics among ALL patients. Although the risk of developing ALL decreases with age in children, it resurfaces in the mid-twenties, with B-ALL being the most common subtype accounting for 80% of acute leukemia cases in children compared to only 20% in adults. Although B-ALL is one of the leading causes of cancer-related death in children, they have a better chance of surviving than adults. The response to treatment is influenced by the type of genomic alterations, indicating that ALL may have different mechanisms in childhood and adulthood.The B-ALL subtype, a prevalent hematologic neoplasm characterized by the uncontrolled proliferation of immature B cells, includes the precursor B-ALL subtype, with the pre-B ALL subtype being the most common form of ALL and predominantly affecting children under the age of 5. Identifying new ALL subtypes with unique gene mutations and rearrangements have improved our understanding of the underlying biology of the disease and has led to the development of targeted therapies that have shown promising results in clinical trials.,25 However, further research is still needed to fully comprehend the genetic and molecular mechanisms underlying ALL and develop more effective therapies for patients.Despite the high incidence of ALL in children, adult ALL patients have a poorer prognosis, possibly due to the differences in genetic alterations between the 2 age groups. In many childhoods, ALL cases do not have a recurrent chromosomal abnormality, despite the possibility of recurrent genetic abnormalities. The genetic propensity of these cases has yet to be fully understood at this time.,28The extent to which a patient responds to treatment for leukemic malignancies is influenced by the type of genomic alterations screened. A study uncovered adult B-ALL’s genetic and molecular architecture, revealing 2 distinct variants that aid in diagnosing patients with Swyer syndrome and Bainbridge. For this review, we will focus specifically on the role of PAX5 mutations in the development of ALL.Precursor B-cell acute lymphoblastic leukemia (B-ALL) has been the focus of intense research in recent years due to the discovery of genetic subtypes with distinct biomarker ramifications. ALL is a complex illness with various risk factors, including genetic predisposition. Distinct recurrent genetic abnormalities can be used to identify ALL phenotypic subgroups; they are crucial for clinical specification and, in some cases, risk grading and precision medicine. It has been discovered that there are over 20 different subtypes of B-ALL, each with its own distinctive risk of relapse. including somatic and germline mutations. It is imperative in the early stages of B cell lineage development, where it plays a role in the commitment and maintenance of B-cells in childhood B-ALL. However, PAX5 is frequently affected by various mutations, including deletions, amplifications, rearrangements, and point mutations.–36 The expression of PAX5 is mainly associated with B lymphocyte maturation, cell survival, motility, and tumor progression, and it varies as cancer progresses. This review will primarily focus on PAX5 mutations and their implications in ALL development.The PAX5 gene, located at 9p13, is a vital transcription factor protein that belongs to the paired box (Pax) family. This family comprises 9 DNA-binding domains crucial in organogenesis and brain development. PAX5 is implicated in the pathogenesis of many cases of ALL,–40 PAX5 plays a crucial role in embryonic development and cell differentiation. It is expressed from the pro-B to the mature B-cell stage in the bone marrow and is downregulated in the final stage of differentiation into plasma cells. PAX5 is the primary target of genetic alterations in B-ALL, with more than a third of patients having mutations that result in downregulation and a defect in DNA-binding activity and expression. Additionally, approximately 2.6% of pediatric B-ALL patients have a rearrangement in PAX5 that fuses it to other genes,,32,35,43 causing B-cell development to be blocked, as seen in PAX5-ETV6 and PAX5-FOXP1 fusions. All fusion genes with PAX are conserved in the paired box, such as PAX5-ETV6, PAX3-FKHR, and PAX8-PPARG. Although almost 80% of childhood cancer cases are curable, many pediatric patients resist therapy, leading to poor outcomes.The PAX5 gene is a paired box (Pax) family member, a domain of 9 highly conserved DNA-binding domains involved in organogenesis and brain development. with a relapse rate of 40.0% compared to 9.6% in pediatrics. Treating ALL remains challenging due to the high risk of relapse shortly after complete remission, which often results in refractory leukemia that is difficult to cure, with a 5-year overall survival rate of approximately 50%. This review will focus on the implications of PAX5 mutations in ALL development, particularly B-ALL. Table Furthermore, adult ALL cases have a poor prognosis, The mutations most commonly associated with PAX5 in ALL, particularly B-ALL, result in downregulation and impair DNA-binding activity and expression, contributing to leukemia.,52 The article underscores how PAX5 is often subjected to deletions, amplifications, rearrangements, and point mutations. The review summarizes the different mutations related to PAX5 and their implications in ALL development. It emphasizes how they lead to the emergence of refractory leukemia that is challenging to cure, with a 5-year overall survival rate of approximately 50%.PAX5 is a critical transcription factor that plays an essential role in B cell lineage development and is also linked to the development of ALL. These rearrangements affect the function of PAX5, resulting in downregulation or a defect in DNA-binding activity and expression, contributing to the development of leukemia. The most frequent rearrangements are with the ETV6 and ZNF521 genes, which lead to ETV6-PAX5 and ZNF521-PAX5 fusion proteins, respectively.,54 These fusion proteins interfere with normal B cell differentiation and maturation, accumulating leukemic cells, and have altered transcriptional activity, leading to the deregulation of target genes involved in B cell development and differentiation. Other rearrangements in PAX5 have also been observed with genes such as BCR, MLL, and FOXP1, which lead to the formation of fusion proteins that affect the function of PAX5 and contribute to the development of leukemia. Detecting rearrangements in PAX5 is crucial for diagnosing and classifying ALL, risk stratification, and treatment selection. The detection of the ETV6-PAX5 fusion protein, for example, is associated with a poor prognosis and a higher risk of relapse, which may require more intensive therapy. Furthermore, the detection of rearrangements in PAX5 provides a potential therapeutic target for the treatment of ALL. Small molecule inhibitors of the ETV6-PAX5 fusion protein have been developed and have shown promising results in preclinical studies.Rearrangements in the PAX5 gene are a common mutation in acute lymphoblastic leukemia (ALL), leading to the fusion of PAX5 with other genes. These translocations disrupt the PAX5 gene and lead to the formation of PAX5 fusion genes with partner genes from other chromosomes. These fusion genes encode chimeric proteins, which can impair normal B-cell development and contribute to leukemogenesis. PAX5 translocations have been reported in approximately 2% to 3% of pediatric and adult ALL cases. The prevalence and prognostic impact of PAX5 translocations vary depending on the specific fusion gene and other genetic alterations present.In ALL, chromosomal translocations involving the PAX5 gene have been identified as significant genetic events.,34,35 A t  translocation is one of the most common PAX5 translocations and is characterized by the presence of a PAX5/ETV6 fusion gene; almost the entire ETV6 transcription factor is fused to the PAX5 paired domain,,53,62,63 one of the essential transcription factors that underlie hematopoiesis as well as B cell development. Interestingly, the abnormalities in the PAX5 gene are often related to dicentric chromosomes, particularly dic , while the exact breakpoints vary widely.,63,65,66 Earlier studies found a fusion between PAX5 and ETV6 in 18 of 19 patients (95%) with dic . The dicentric  translocation usually exists in B-ALL,65 and is closely correlated with PAX5-ETV6 fusion; however, it is also present in other chromosomal alterations, such as t  that comprise ETV6-RUNX1, demonstrating that these actions are coordinated.,67PAX5 is often involved in translocations along with transcription factors, structural proteins, kinases, and other genes.,34 PAX5-ETV6 and PAX5-FOXP1 are examples of chimeric PAX5 fusion proteins, which are multi-domain transcription factors. In addition to their DNA-binding domains , both proteins contain an oligomerization motif (pointed domain for ETV6 or a coiled-coil domain for FOXP1).,63 Accordingly, PAX5-ETV6 and other PAX5 fused proteins oligomerize.,68 Various PAX5 fusion proteins, including PAX5-ETV6 and PAX5-FOXP1, have been reported to suppress the expression levels of wild-type PAX5 in temporal transfection experiments.,43,67,69While PAX5-FOXP1 is found in fewer B-ALL cases, it is still a recurrent translocation that connects almost the entire FOXP1 transcription factor sequence to the N-terminal PAX5 sequence.https://www.ncbi.nlm.nih.gov/gene/2120). In human B-ALLs, PAX5 appears to function as a haploinsufficient tumor suppressor. There needs to be more information about the PAX5 mutations that result from the fusion of PAX5 and other genes.,35ETV6 belongs to the ETS family of transcription factors. Its protein contains a pointed N-terminal domain that interacts with other proteins or with itself and a-terminal DNA-binding domain. This gene is shared in a chromosomal rearrangement that causes leukemia , and he has pancytopenia, 0.9 × 109/L white blood cells with 30 percent of blasts, he was diagnosed with B-cell ALL (B-ALL). In 8 metaphases, karyotyping reveals 45, XY, 12, and dic  . The CMA result demonstrated the location and the deletion range, 37 Mbp of deletion on chromosome 9, along with discontinuous 12 and 14 Mbp deletions on chromosome 12. CMA result of fusion detection, a fusion between 3′ of exon 4 of PAX 5 and 5′ of exon 3 of ETV 6 , a motif -PAX5/ETV6 as dic  . Therefore, it is clear that dic , described as a chromosomal mutation primarily found in pre-B hematopoietic malignancies, occurs due to PAX5-ETV6 fusion.In another study, 2 patients were diagnosed with ALL L1; one was treated with the ALL-BFM2000 protocol, and the second with the ALL-BFM86 protocol. The cytogenetic test revealed t   in the first patient and dic   in the second. The abnormalities in 12p influence the ETV6 gene, so the FISH test is used to examine ETV6. The 3′–end of the gene persisted on the dic  chromosome, but the 5′-end was removed. The breakpoints on the cytogenetic test indicated that ETV6 may have presumably fused with PAX5. PAX5’s 5′ end has been found on the dic  chromosome, but the 3′ end has been removed.https://www.ncbi.nlm.nih.gov/gene/2006).ELN encodes one of the elastic fiber proteins, which give tissues such as skin, lungs, and blood vessels elasticity. Elastokines are the products of ELN protein degradation. They induce monocyte proliferation and can promote cancer to progress  . The FISH test detected the arrangement in PAX5; results reveal a breakpoint between exons 7 and 10. So PAX5 is fused with another gene. Sequencing demonstrated that the partner gene of PAX5 is ELN. RQ-PCR was done on DG75 cells to measure the effect of PAX5-ELN on other endogenous genes, revealing that PAX5-ELN down-regulated LEF1, BLNK, and MB1 and did not affect the transcription of BLK and CD19. PAX5-ELN competes with PAX5, so it may interfere with B-cell differentiation, closely linked to PAX5 expression.PAX5-ELN and PAX5 were both detected in the nucleus by confocal microscopy. The results of CHIP experiments clearly showed that PAX5-ELN could bind BLK, CD19, and wild-type PAX5 sequences, suggesting a dominant-negative activity. HeLa cells co-transfected with a luc-CD19 construct, pcDNA3-PAX5, and pcDNA3-PAX5-ELN showed a decline in PAX5-driven CD19 transcription compared to the amount of pcDNA3-PAX5-ELN transfected. Consequently, PAX5-ELN could block PAX5-dependent transactivation by interacting with PAX5-binding sites. Several studies indicate that FOX proteins are critical for immune control, regulating the survival of lymphocytes to the development of thymocytes. Furthermore, the FOX transcription factors play a role in carcinogenesis by chromosomal translocation, retroviral integration, gene amplification, and transcriptional regulation. FOXP1 is confirmed by gene expression and immunophenotypic studies; it is regulated on normal B cells but overexpressed on diffuse large B-cell lymphoma subgroups. Mutations in FOXP1 cause a disturbance in the development of B cells because it plays an essential role in regulating B lymphopoiesis.FOXP1 (Forkhead box protein P1) is one of a group of transcription factors called FOX; their DNA-binding domain is named the forkhead domain or the winged-helix. In 2/6 and 4/4 metaphases, respectively, and in 164/200 and 175/200 interphase nuclei, his FISH result was ish t    and ish t   . The forkhead box P1(FOXP1) gene was fused with the paired box 5 (PAX5) gene.A translocation t   was found in a man with B-cell acute lymphoblastic leukemia (B-ALL). His blood count was 37.230 × 109/L with leukocytosis and 60.3% blasts, his LDH level was (1710 IU/L),  for thrombocytopenia, and his Hemoglobin was normal. A sample from his bone marrow revealed TdT, CD10, CD20, and cyCD79a were positive, while cym, CD3, and myeloperoxidase were negative. identified.Another study used a fusion of PAX5 and FOXP1 at exons 6 and 7 to ensure chimeric transcription in 2 patients with t  , which RCA-RACE and FISHThe 9p and 3′ portions of PAX5 were deleted in 5 patients with B-progenitor cells. Two of them have a fusion of exon 6 and exon 7 of PAX5 and (FOXP1), respectively, and these results were ensured by RT-PCR, FISH, and sequencing analyses. Mutated Pax5 decreased the activation of transcription, and the transaction process to increase PAX5-FOXP1 and a constant amount of PAX5 wild type showed the fusion proteins blocked the transcription process of PAX5.,67 The translocation in the other 2 females was examined using RCA-RACE. The fusion of PAX5 and FOXP1 inhibits PAX5 activity, which may lead to leukemogenesis by preventing hematopoietic cells from converting into mature B cells.,35,67Only 4 cases of PAX5-FOXP1 translocation have been reported so far. Translocation in 2 was examined by a single nucleotide polymorphism array.https://www.ncbi.nlm.nih.gov/gene/5371).This gene encodes a protein that belongs to the tripartite motif family. It is localized in nuclear bodies, serving as a transcription factor and tumor suppressor. In response to oncogenic signals, this gene regulates p53. It is known to be involved in translocations with the retinoic acid receptor alpha gene, which is related to acute promyelocytic leukemia  in the first and a t   in the second. FISH and hybridization ensured that PAX5 and PML were fused. Accordingly, the karyotype was refined to be t   in the first patient, and t   in the second. This fusion inhibited PAX5 transcription in the luciferase assay and suppressed the expression of PAX5. Because PAX5 is essential for B-cell differentiation, this dominant-negative effect may block differentiation in PAX5-positive ALL. Furthermore, PAX5–PML inhibited the simulation of PML, interrupted the PML nuclear bodies, and induced apoptosis resistance in HeLa cells. Disruption in PML nuclear bodies, essential for lymphocyte apoptosis, caused acute promyelocytic leukemia cells to survive by binding to PML-RAR. So, this obstructive effect on PML induces cells of PAX5–PML-positive ALL to survive. Because of this strong negative effect on PML, PAX5-PML + ALL cells will be more likely to persist  has shed light on the genetic basis of this deadly disease. The PAX5 gene, which is essential for B-cell development, plays a critical role in the pathogenesis of ALL when it becomes fused with other genes, resulting in the formation of abnormal proteins that disrupt normal cellular function. Identifying specific PAX5 fusion partners and their clinical implications has opened up new avenues for developing targeted therapies for ALL. The prognosis of ALL has improved over the years with advances in chemotherapy, stem cell transplantation, and supportive care. However, the prognosis remains poor for patients who relapse or are refractory to treatment, especially those with high-risk genetic features. The discovery of PAX5 fusion has provided a better understanding of the underlying molecular mechanisms of ALL and has the potential to improve clinical outcomes for patients with this disease. Several studies have shown that patients with PAX5 fusions have a distinct clinical and genetic profile compared to those without PAX5 fusions. They tend to be younger, have higher white blood cell counts, and are more likely to have additional genetic abnormalities. The identification of these features can help in risk stratification and treatment decision-making. For example, patients with PAX5 fusions have been shown to respond better to glucocorticoid therapy, which is a cornerstone of ALL treatments.Additionally, targeted therapies such as tyrosine kinase inhibitors and immunotherapy may benefit patients with PAX5 fusions. The development of targeted therapies for PAX5 fusions in ALL is an active area of research. Several promising drugs are in preclinical and clinical development, including inhibitors of the BCR-ABL1 and JAK-STAT signaling pathways. These therapies have shown promising results in preclinical studies and early-phase clinical trials and may provide an effective and less toxic alternative to conventional chemotherapy. In conclusion, the discovery of PAX5 fusion in ALL has provided valuable insights into the genetic basis of this disease and has the potential to improve clinical outcomes for patients. Identifying specific PAX5 fusion partners and their clinical implications has opened up new avenues for targeted therapy development, which holds great promise for the future of ALL treatment. Further research is needed to fully understand the molecular mechanisms of PAX5 fusion and develop more effective and less toxic treatments for this devastating disease.We thank the Egypt Scholar Foundation and Prof Ahmed Abdel Aziz Bayoumi in the Faculty of Science at Cairo University for their assistance and advice.Resources: Fatma Mohamed Fouad.Supervision: Jehane I. Eid.Validation: Fatma Mohamed Fouad.Visualization: Fatma Mohamed Fouad.Writing – original draft: Fatma Mohamed Fouad.Writing – review & editing: Jehane I. Eid.


As we have learned, chromatin modifications, including histone modifications and DNA methylation, play a key role in plant development . HoweverFLC expression by autonomous pathway genes includes histone modifications, recent advances indicate that this process is much more complex. It has been shown that the precise control of FLC expression additionally involves chromatin architecture, RNA polymerase pausing, and ncRNA-mediated gene silencing. The review by Kyung et al. discusses how these novel mechanisms coupled with histone modifications may lead to the repression of FLC expression and provides the reader with a comprehensive review of autonomous pathway gene-mediated FLC repression via epigenetic regulations.Flowering locus C(FLC)is a major determinant of flowering in Arabidopsis. Whereas the repression of C. elegans, they have been tightly connected to organisms’ development. Although there are different biogenesis mechanisms between plants and other organisms, miRNAs have been confirmed to play a key role in plant development, e.g., miRNA156/172 regulating developmental timing. For instance, pre-miRNAs that are transcribed from MIR genes by DNA-dependent RNA polymerase mediate MIR promoter accessibility and MIR gene transcription. In addition to this role, epigenetic factors also regulate miRNA biogenesis and abundance. Recent evidence has shown that a key component Serrate (SE) of miRNA processing complex can directly interact with CHROMATIN REMODELLER 2 (CHR2) and unwind pre-miRNA structure, thus preventing miRNA biogenesis and accumulation. However, CHR2 also can function as a positive regulator of MIR gene transcription via its chromatin remodeling activity. The different and even opposite roles of CHR2 in miRNA biogenesis embody the complexity of epigenetic regulations. This and other detailed advances in miRNA biogenesis and stability can be found in the review article by Zhang et al..Since the discovery of miRNAs in Inácio et al..Secondary growth mediated by secondary meristems is crucial for plant radial thickening and plant axes strengthening. Whereas vascular cambium leads to secondary xylem and phloem, cork cambium or phellogen produce the periderm, with an important role to protect plants from insects, diseases, and the harmful effects of climate change. Secondary growth mediated by vascular cambium such as during xylogenesis, dormancy-activation periods of cambium, and secondary tissue regeneration after injury are under epigenetic regulations, involving histone modifications (H3K4me3), DNA methylation, chromatin remodeling, and miRNA-mediated DNA methylation. During periderm formation, phellogen-mediated cell division, differentiation, and regeneration are regulated by various epigenetic modifications, including increased DNA methylation followed by chromatin condensation, and H3K4me3 enrichment to activate genes involved in secondary cell wall deposition and programmed cell death. In addition, miRNAs targeting histone modifiers cause alterations in the histone modification landscapes and also mediate periderm differentiation and formation. More in-detailed epigenetic effects on plant secondary growth can be found in the review paper by FLC expression, HDA9 targets AGL19, and HDA19 regulates photoperiod genes. In the Research Topic, Lee et al. show that HDA9 represses GIGANTEA expression under short-day conditions, thus stimulating hypocotyl cell elongation.Histone modifications play a key role in regulating developmental genes, and RPD3-like histone deacetylases, e.g., HDA6, HDA9, HDA19, etc., can form conserved SIN3-type histone deacetylase complexes to regulate plant responses to stresses and developmental cues. Among them, HDA6 maintains heterochromatin status by preventing DNA demethylation at heterochromatin regions through deacetylating H3K18ac, a crucial mark for DNA demethylases . AlthougDelay of germination 1 (DOG1) has been described as controlling seed dormancy by converging with the ABA signaling pathway to tightly repress seed germination. Previous studies with different Arabidopsis ecotypes adapted to summer (Bur) and winter (Cvi) seasons found that histone modification H3K4me3 remains stable during dormancy, and as dormancy declines, H3K4me3 level decreases. During the release of dormancy, H3K27me3 repressive mark slowly accumulated along DOG1. In this Research Topic, Han et al. show that HD2A and HD2B are recruited by HSI2 and HSL1 to downregulate DOG1 expression and to release seed dormancy. These results show that various epigenetic modifications coordinate together to make a fine tune for seed dormancy and germination.Seed dormancy is not only an important developmental process but also affects plant survival and adaptation to adverse habitats. DOG1 gene and HDA19-mediated repression of GIGANTEA. In addition, this Research Topic brings the latest epigenetic advances on periderm development as well as the chromatin architecture effects on FLC expression and epigenetic interaction with miRNA biogenesis and stability. Given the fact that histone deacetylases interact with epigenetic machinery, such as chromatin remodeling complexes and numerous transcription factors involved in important regulatory processes, the further exploration of this field may lead to the deciphering of not yet recognized precise regulatory mechanisms controlling gene expression in the context of the response to changing environmental conditions. Thus, the articles presented in this Research Topic provide qualified and valuable knowledge for the epigenetic community.In the Research Topic, readers will find how histone modifications affect developmental gene expression and regulation, e.g., histone acetylation on the All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Candida albicans as they are required for adhesion to and invasion of host cells, and ultimately deep-tissue dissemination. Hyphae also secrete the peptide toxin candidalysin, which is crucial for destruction of host cell membranes. The peptide is derived from a precursor protein encoded by the gene ECE1 which is strongly induced during hyphal growth. Previous studies revealed a very complex regulation of this gene involving several transcription factors. However, the promoter of the gene is still not characterized. Here, we present a functional analysis of the intergenic region upstream of the ECE1 gene. Rapid amplification of cDNA ends (RACE)-PCR was performed to identify the 5′ untranslated region, which has a size of 49 bp regardless of the hyphae-inducing condition. By using green fluorescent protein (GFP) reporter constructs we further defined a minimal promoter length of 1,500 bp which was verified by RT-qPCR. Finally, we identified the TATA element required for the expression of the gene. It is located 106 to 109 bp upstream of the ECE1 start codon. Our results illustrate that despite a very short 5′ UTR, a relatively long promoter is required to secure ECE1 transcription, indicating a complex regulatory machinery tightly controlling the expression of the gene.The formation of hyphae is a key virulence attribute of IMPORTANCE In recent years it was shown that secretion of the toxic peptide candidalysin from hyphae of the major human fungal pathogen Candida albicans contributes heavily to its virulence. The peptide is derived from a precursor protein which is encoded by the ECE1 gene whose transcription is known to be closely associated with formation of hyphae. Here, we used a GFP reporter system to determine the length of the ECE1 promoter and were able to show that it has a minimal size of 1,500 bp. Surprisingly, the gene has a very short 5′ UTR of only 49 bp. In accordance with this, the TATA element required for transcription is located 106 to 109 bp upstream of the start codon. This indicates that ECE1 expression is controlled by a very long promoter allowing a complex network of transcription factors to contribute to the gene’s regulation. Candida albicans  the exact length of the promoters is unknown  can inhibit translation of the mRNA  reporter constructs we defined a minimal promoter required for full-level expression of ECE1. Further we utilized 5′ rapid amplification of cDNA ends (RACE)-PCR to identify the length of the 5′ UTR and located the central TATA regulatory element.Here, we present a functional analysis of the 5′-intergenic region of the ECE1 is located on chromosome 4. The current assembly of the of the C. albicans genome shows three small open reading frames (ORFs) upstream of ECE1. All of them have a size of approximately 500 bp and are annotated as C4_03480C (588 bp), C4_03490C (525 bp), and C4_03500C (552 bp). A gene with verified function, HWP2, is located upstream of them  transfer from yeast extract-peptone-dextrose (YPD) to RPMI 1640 medium. The 5′ RACE-PCR was performed after 60 min of incubation. A PCR product corresponding to the 5′ UTR was neither observed in YPD nor synthetically defined glucose medium (SDG) medium, which was expected as C. albicans exclusively grows in yeast form in both media .The fusion PCR products containing either the TATA box 1 or the TATA box 2 mutations were finally restricted with C. albicans SC5314 by using the established lithium acetate method  which contained 10% human serum if required. Cells were then grown for 1 or 2 h at 37°C. Afterwards, total RNA was isolated from the harvested cells as previously described  and gene-specific primers for ECE1 . Oligonucleotide primer GSP3 contains a XhoI restriction site for the subsequent cloning of the final PCR product into pSK plasmid, allowing Sanger sequencing of the PCR product and identification of the 5′ UTR sequence.The size of the 5′ untranslated region of for ECE1 . RACE-PCFluorescence microscopy was performed with a Nikon Eclipse Ni microscope . Differential interference contrast (DIC) illumination time was 200 ms and for the GFP channel it was 2,000 ms. The same illumination times were used for all samples to guarantee comparable GFP signals.


Rosa roxburghii fruit are still unclear. We utilized transcriptomics and metabolomics to investigate genes related to sugar and organic acid metabolism in Rosa roxburghii. Metabolomics data revealed that sucrose, glucose and fructose were the primary sugars, whereas citric acid and malic acid were the primary organic acids in Rosa roxburghii fruit. We constructed the metabolic pathways of major sugars and organic acids in Rosa roxburghii and identified five key genes involved in sugar and organic acid synthesis. In addition, we identified a module containing 132 transcription factors that was significantly associated with sucrose, citric acid and malic acid. Based on quantitative polymerase chain reaction (qPCR), we identified 13 transcription factors involved in sugar and organic acid metabolism, including the transcription factor RrANL2 and the sucrose synthase gene RrSUS3. Further yeast one-hybrid and dual luciferase assays showed that RrANL2 could bind to the promoter of RrSUS3 to increase its expression. These results provide new insights into the metabolism of sugars and organic acids in Rosa roxburghii fruit.Sugars and organic acids significantly impact fruit sensory quality, but their accumulation patterns and regulatory mechanisms during the development of  Sweetness is an important factor that impacts the quality of most fruits . The totSourness is another factor that determines the quality and ripeness of fruit . The sweRosa roxburghii is an economically important fruit tree that is native to China, where it is mainly distributed in southwest regions such as Guizhou Province, Yunnan Province and Sichuan Province. Rosa roxburghii fruits have recently been found to contain very high vitamin C content , in addition to other bioactive molecules such as vitamins, polyphenols, flavonoids, organic acids and Superoxide dismutase [Rosa roxburghii fruit has a slightly sour and bitter taste, reducing its potential consumer appeal. At present, Rosa roxburghii fruit is primarily utilized in an array of processed products, including dry fruit, canned fruit and fruit drinks [Rosa roxburghii fruit, a better understanding of the dynamic changes of sugar and organic acid content during the course of its development is required. Although the metabolic processes associated with sugars and organic acids are well known in many plant species, they are poorly understood during Rosa roxburghii fruit development. A better understanding of these processes can lead to the development of Rosa roxburghii fruit with better consumer appeal, while retaining its beneficial nutritional qualities.ismutase ,16. Despt drinks . To imprRosa roxburghii development were analyzed through metabolomic and transcriptomic analyses. Metabolic pathways for sugars and organic acids were constructed, and several key enzymes regulating sugar and organic acid synthesis were identified. We also conducted a focused investigation of the role of transcription factors in the accumulation of sugars and organic acids in Rosa roxburghii. Application of weighted gene co-expression analysis (WGCNA) led to the creation of modules that correlated transcription factors with sugars and organic acids. Within the modules significantly associated with sugar and organic acid accumulation, we identified many transcription factors and selected the differentially expressed transcription factors and key enzymes regulating sugar and organic acid synthesis in Rosa roxburghii for quantitative polymerase chain reaction validation. The regulatory relationships of transcription factors with sugar and organic acid synthesis enzymes were also verified by yeast one-hybrid and dual luciferase assays. This study provides a reference for understanding the mechanisms of sugar and organic acid accumulation in Rosa roxburghii fruits which can be applied to genetic breeding and quality improvement.In this study, the dynamics of sugars and organic acids during Rosa roxburghii fruits, we used transcriptomics combined with metabolomics to assay gene expression and metabolite content at five different stages of Rosa roxburghii fruit development  was only highly expressed in S1 and S2. The A-N-INVG gene family members evm.TU.chr3.3618, evm.TU.chr5.1377 and evm.TU.chr6.3991 were highly expressed at the fruit ripening stage, and evm.TU.chr4.5684 and evm.TU.chr7.916 were highly expressed at the early stages of fruit development. In addition, members of the AGPase gene family were downregulated over the course of fruit development, reducing the conversion of sugar to starch. However, members of the AMY gene family were all upregulated during development, enhancing the degradation of starch to glucose. These findings indicate that sugar metabolism is regulated by multiple genes working together during Rosa roxburghii fruit development.The sugar and organic acid content of fruit are the two most important factors impacting its flavor. Having already identified the primary sugars and organic acids in pathways . Based ofamilies . SUS genRosa roxburghii genome , evm.TU.chr6.3991 (A-N-INVC), evm.TU.chr4.5028 (AMY1) and evm.TU.chr3.4947 (AMY3) consistently increased during fruit development, with 1.58, 2.33, 7.08 and 2.45-fold increases compared with the S1 stage. There were also 4.68 and 8.61-fold increases in the organic acid metabolic genes CS2 (evm.TU.chr1.2040) and ME1 (evm.TU.chr1.7029), respectively. We also performed qPCR analysis on the aforementioned genes, and the results were comparable to those of RNA-seq analysis. Additionally, all surveyed genes, except AMY3, were significantly expressed at the S5 stage , WRKY (4), trihelix (4), bZIP (4), ERF (3) and others  and evm.TU.chr1.2040 (RrCS2), whereas RrANL2 and RrGBF3 bound to the promoter of evm.TU.chr4.1713 (RrSUS3) ((RrSUS3) A. RrANL2 and RrGBF3) and RrSUS, we performed dual luciferase assays. This analysis showed that RrANL2 could enhance the fluorescence signal of pro-RrSUS::LUC, whereas RrGBF3 had no effect on the fluorescence signal of pro-RrSUS::LUC  belongs to the HD-ZIP IV subfamily [ANL2 has been shown to be involved in sucrose metabolism in plants, where it impacts the level of the sucrose transporter protein AtSUC1 in Arabidopsis thaliana [RrANL2 was an upstream regulator of RrSUS3, which provides new insights into the regulation of sugar metabolism in Rosa roxburghii fruit.We analyzed the expression of 14 transcription factors during wth rate ,33,34,35ubfamily ,37,38. Athaliana . AdditioRosa roxburghii fruit development. During this process, genes associated with sugar and organic acid pathways were differentially expressed, with biosynthetic genes primarily increasing in expression during fruit development. Additionally, numerous transcription factors that may control sugar and organic acid metabolism in Rosa roxburghii were identified by WGCNA. Moreover, the transcription factor RrANL2 was found to regulate the expression of RrSUS3, a key gene in sucrose synthesis. Taken together, these results partially elucidate the metabolism of sugars and organic acids in Rosa roxburghii and provide a biological basis for understanding their accumulation in Rosa roxburghii fruit. In this study, we integrated transcriptomic and metabolomic techniques to investigate the accumulation patterns of sugars and organic acids during Rosa roxburghii material used in this study was collected from the Baima Experimental Base of Nanjing Agricultural University, Jiangsu Province, China. Three biological replicates were collected for each sample 10, 40, 70, 100 and 130 days after flowering and named S1, S2, S3, S4 and S5. Samples were frozen in liquid nitrogen after collection and stored in a −80 °C freezer for later use.The http://www.kegg.jp/kegg/compound/, accessed on 11 February 2022) and KEGG pathway database . Inclusions of samples from the five fruit stages were dissolved in 70% methanol and left overnight at 4 °C. After centrifugation at 10,000 rpm for 10 min, the supernatant was analyzed using a UPLC-ESI-MS/MS system . Metabolites were considered differentially accumulated when they had an absolute Log2FC (fold change) ≥ 1 with a VIP ≥ 1. Metabolites were annotated and examined for enrichment using the KEGG compound database . RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer , and RNA integrity was assessed using an RNA Nano 6000 Assay Kit from the Agilent Bioanalyzer 2100 system . After quality assessment, 1 µg of RNA was utilized for RNA-seq library construction. Paired-end RNA-seq reads were generated using the Illumina NovaSeq 6000 platform. Raw RNA-seq data were then filtered with fastp  and mapprameters . We calcRosa roxburghii fruit development, we identified transcription factors in the Rosa roxburghii genome by BLASTP [Rosa roxburghii fruit development [To understand the effects of transcription factors on sugars and organic acids during y BLASTP  using aly BLASTP . Additioelopment . Using gRosa roxburghii fruit using a Foregene Plant Total RNA Extraction kit . RNA was subsequently reverse-transcribed into cDNA by the PrimeScript RT Reagent Kit . The relative expression patterns of target genes during Rosa roxburghii fruit development were analyzed with the Hieff UNICON Universal Blue qPCR SYBR Green Master Mix . We utilized the expression of Rosa roxburghii RrUBQ as a reference gene for calculating relative expression by the 2−∆Ct method and determined statistical significance using SPSS22. All primers used in this analysis are listed in Total RNA was extracted from five stages of Rosa roxburghii fruit using the Foregene Total Plant RNA Extraction Kit and Foregene Plant DNA Extraction Kit, respectively. cDNA was obtained by reverse transcription of RNA using the PrimeScript RT Reagent Kit (TaKaRa). The transcription factor sequences were amplified by PrimeSTAR HS DNA Polymerase (TaKaRa) and ligated into pGADT7 and PJX001 vectors. Additionally, the promoter sequence of each target gene was amplified from Rosa roxburghii genomic DNA and ligated into pHIS2.1 and pGreenII 0800-LUC vectors. All primers used in this analysis are listed in We obtained total RNA and genomic DNA from 2O and their concentration was adjusted to an OD600 of 0.05. Next, 5 µL of the yeast strain suspension was aspirated and transferred to SD/-Leu/-Trp/-His liquid medium  containing different concentrations of 3-AT  and grown for 4 days at 30 °C.Yeast one-hybrid assays were used to determine the binding relationships between transcription factors and promoters. The pHIS2.1 vector carrying the transcription factor pGADT7 vector and the pHIS2.1 vector carrying the promoter sequence were transformed into the Y187 yeast strain by a modified lithium acetate method. The yeast strain was subsequently plated on SD-Leu-Trp solid agar medium  and incubated at 30 °C. After 3 days, single colonies of yeast were picked, suspended in ddHThe regulatory relationship between transcription factors and promoters was further verified using dual luciferase assays. PJX001 vectors carrying transcription factors and pGreenII 0800-LUC vectors carrying promoter sequences were transformed into the Agrobacterium tumefaciens GV3101 receptor. The different combinations of transcription factors and promoters were then injected into tobacco leaves in a 9:1 ratio. After 3 days, tobacco leaves were assessed for LUC activity to determine the interactions between different promoters and transcription factors.


The stabilisation of G4s with small molecule ligands has emerged as a technique to regulate gene expression and as a potential therapeutic approach for human diseases. Here, we demonstrate that ligand stabilisation of G4s causes altered chromatin accessibility dependent on the targeting specificity of the molecule. In particular, stabilisation of a target G4 using the highly specific GTC365 ligand resulted in differential accessibility of 61 genomic regions, while the broad-targeting G4 ligand, GQC-05, stabilised many G4s and induced a global shift towards increased accessibility of gene promoter regions. Interestingly, while we observed distinct effects of each ligand on RNA expression levels and the induction of DNA double-stranded breaks, both ligands modified DNA damage response pathways. Our work represents the dual possibility of G4-stabilising ligands for specific or global chromatin modulation  DNA G-quadruplex-stabilising ligands can induce global or specific changes in chromatin accessibility and the transcriptome depending on the targeting specificity of the molecule. The principal factors governing this reorganisation in turn cause gene activation or silencing by regulating accessibility of gene promoters. Epigenetic modifications, mutations, and DNA secondary structures have all been investigated for their role in influencing chromatin accessibility and gene transcription.1–3 Of the various secondary structures that have been associated with chromatin accessibility and nuclear processes, G-quadruplexes (G4s) are particularly interesting as they have been identified as therapeutic targets for diseases including cancer4,5 and neurodegenerative disorders.6 G4s are sequence-specific structures that consist of stacked G-tetrads, each composed of four Hoogsteen hydrogen-bonded guanines in a planar arrangement.7 Unlike epigenetic and point mutations in the genome, which are heritable and stable modifications,8 G4 formation is dynamic and transitory. The propensity for DNA G4 formation in the human genome is very high, with over 10 000 G4-forming regions in chromatin and over 700 000 G4-forming regions identified in naked genomic DNA.9,10Nuclear processes such as gene expression are influenced by a range of mechanisms, including the arrangement of nuclear architecture. The reorganisation of nuclear architecture 5,10–18 Targeted interaction and stabilisation of specific G4s using small molecules has been widely accepted as a therapeutic strategy.15,16,19–22G4 structures are enriched in gene promoters and nuclease hypersensitive regions, coincide with binding sites for transcription factors and DNA methyltransferases, and are implicated in transcriptional regulation, replication, and genomic instability.23,24 Chromatin accessibility is required for transcription, and remodelling of chromatin is central to the DNA damage response to facilitate access of repair factors for removal of the lesion.25–28 Recently, G4 formation has been correlated with DNA damage repair and gene activation.18,29–31 Thus, G4s may contribute to the modulation of chromatin organisation, as reported by Zyner et al.32 In this work, we show that small molecule-induced G4 stabilisation can be used as a strategy to regulate chromatin remodelling in human cells. In particular, we show that G4 stabilisation and chromatin remodelling are interdependent phenomena. Enhanced chromatin remodelling of specific gene promoter regions was observed for the case of targeted G4 stabilisation using a ligand specific for the G4 in the promoter region of human telomerase reverse transcriptase (hTERT). On the other hand, broad-targeted, genome-wide stabilisation of G4s causes a global shift towards increased promoter accessibility, although few regions are consistently altered. Our findings using Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) and RNA-sequencing (RNA-seq) analysis coupled with G4 formation predictions in MCF-7 human breast adenocarcinoma cells reveal that G4s are key regulators of chromatin remodelling in human cells.Stabilisation of G4s using small molecules has been identified as an important approach to regulate gene expression, but G4 stabilisation can increase DNA damage in cells if G4s are not efficiently regulated.33 On the other hand, GTC365  BG4 antibody,36 and DNA damage identified using an antibody against γH2AX, a marker for DNA double-strand breaks.37 The broad-targeting G4 ligand, GQC-05, resulted in a significant increase in both γH2AX and BG4 foci counts, while the hTERT G4-targeting ligand, GTC365, resulted in a smaller increase in BG4 foci and no significant change in DNA damage , defined as regions with false discovery rate (FDR) < 0.1 to allow for the largest range of genomic interactions to be identified. Of these 61 DARs, 22 were located in gene promoter regions. On the other hand, GQC-05 treatment resulted in only 5 DARs  and GTC365 (46.2%) . This isy 5 DARs . This su38 The pG4 and ATAC-seq datasets were intersected to identify pG4s within promoter regions exhibiting differential accessibility  for at least 21 out of 22 randomly selected regions to contain a pG4. The single DAR without a pG4 has increased accessibility. In contrast, 25 out of 39 non-promoter DARs had increased accessibility and 14 were reduced  within the genome were identified using the G4Hunter algorithm.sibility  and S3†.2 FC| > 2 to identify genes that are maximally altered by the ligands. GQC-05 did not cause any significant differential expression, similar to the low number of DARs induced by this broad-targeting G4 ligand. GTC365 treatment resulted in 162 DEGs  defined using the threshold FDR < 0.05 and |log162 DEGs , with 40 Table S2. Interes33,42 However, for 72 h treatment as in this work, GQC-05 has relatively high toxicity and was therefore used at a low concentration to maintain cell viability. At this concentration, it is likely that the number of G4-ligand complexes formed is insufficient to stabilise all possible targets in each cell, with over 700 000 genomic G4-forming sequences having been experimentally validated.9 Additionally, Brown et al. demonstrated that for the MYC promoter G4, for which GQC-05 has the highest reported affinity, the GQC-05 : G4 stoichiometry is 2 : 1, indicative of two ligand binding sites per G4 for complete stabilisation.33 The binding constants for these two binding sites are 0.1 μM and 1.43 μM, so both the IC50 (0.51 μM) and the concentration used in this study (0.3 μM) will not result in consistent G4 stabilisation across cells. Consequently, different subsets of G4s may be stabilised in each cell, resulting in a reduced average effect on the genomic architecture and gene expression that may be better identified via single-cell studies rather than bulk RNA- or ATAC-seq. While higher doses of GQC-05 have been shown to induce transcriptomic modifications over short treatment periods,33,42 GQC-05 cytotoxicity varies significantly between cell types, and the lower dose treatments have different effects on gene expression in different cell lines.33,43The lack of differential gene expression induced by GQC-05 was unexpected as it stabilises a wide range of G4s within cells and has been associated with differential gene expression over shorter time periods and at higher concentrations.via RNA-seq, ranked by their expression levels. Many pathways were identified as differentially enriched by the two ligands, including several associated with DNA and RNA processes  pathway analysis and Gene Ontology (GO) enrichment analysis was conducted on genes containing ATAC-seq peaks in the promoter region, and Gene Set Enrichment Analysis (GSEA) performed on all genes identified rocesses . Of note48 Similar motifs were found to be enriched in each dataset, including the control  binding sites, we conducted HOMER analysis on the ATAC-seq peaks to identify enriched TF binding motifs.In this work we have demonstrated that treatment of MCF-7 cells with G4-stabilising ligands alters the genomic landscape, transcriptome, and DNA damage response. Modifications in chromatin accessibility are observed to correspond to the extent of G4 stabilisation and targeting specificity of the G4 ligands. GTC365, which has been reported to target a specific G4, induced consistent, significant differential accessibility of certain regions of the genome, while the broad-targeting G4 ligand, GQC-05, caused general shifts in the chromatin landscape that increased accessibility of gene promoter regions. We identified novel GTC365-induced decreases in promoter accessibility that agrees with the documented transcriptional repression associated with ligand-induced G4-stabilisation. Unlike GQC-05, GTC365 caused significant differential gene expression with the majority of altered genes being downregulated. This represents the dual possibility of small molecule G4-stabilising ligands as therapeutic agents to target G4s in diseases, inducing either specific or global effects depending on the design of the molecule.The raw data reported in this paper are available at the NCBI GEO repository. Processed data files are included in the ESI.†A. O. and J. J. K. performed cell culture experiments and A. O. prepared samples for sequencing. N. B. L. conducted sequencing analysis and bioinformatic predictions. N. M. S., K. S. I. and C. W. E. developed the concept. All authors discussed and analysed results and contributed to the manuscript.There are no conflicts to declare.SC-014-D3SC02533K-s001SC-014-D3SC02533K-s002SC-014-D3SC02533K-s003SC-014-D3SC02533K-s004SC-014-D3SC02533K-s005


During embryonic development, stem cells undergo the differentiation process so that they can specialize for different functions within the organism. Complex programs of gene transcription are crucial for this process to happen. Epigenetic modifications and the architecture of chromatin in the nucleus, through the formation of specific regions of active as well as inactive chromatin, allow the coordinated regulation of the genes for each cell fate. In this mini-review, we discuss the current knowledge regarding the regulation of three-dimensional chromatin structure during neuronal differentiation. We also focus on the role the nuclear lamina plays in neurogenesis to ensure the tethering of the chromatin to the nuclear envelope. The epigenome acts as a film director, assigning different roles to each cell type by defining when and what genes are accessible to transcription factors to regulate their expression. Additionally, the epigenome contributes to gene expression through the modification of the three-dimensional organization of the genetic material (chromatin). The epigenome regulates the DNA via DNA methylation on the cytosine of CpG dinucleotides and post-translational modifications of the histone proteins, but also via the activity of non-coding RNA and chromatin remodellers [The DNA sequence of all cells in the body is the same even though the cells do not play the same function. Each cell type is characterized by a precise orchestration of gene expression for the production of all proteins necessary for specific cellular function. Hence, not all genes are active in all cells at any given time, and their expression is tightly regulated by the epigenome. The Greek preposition “odellers . The effodellers ,3 and geodellers . Indeed,odellers ,6. DNA modellers ,8. Histoodellers ,10. Impoodellers ,12. Chroodellers . Membersodellers . On the odellers .Long non-coding RNAs (lncRNAs) can modulate chromatin structure and regulate the transcription of adjacent and distant genes, but can also affect RNA splicing, stability and translation ,17. LMNA T10I in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) causes increased expression of alternative fate genes , which are normally repressed as bound to the nuclear lamina [Malhas and colleagues demonstrated that depletion of the C-terminus of Lamin B1 causes increased susceptibility to ROS formation in a model of mouse embryonic fibroblasts (Lmnb1Δ/Δ cells) [Drosophila Melanogaster embryos, the Twist factor, important for Drosophila development, increased both in terms of mRNA and protein due to the nuclear accumulation of Armadillo protein [Engler and colleagues demonstrated that human mesenchymal stem cells (MSCs) differentiated towards the brain, muscle or bones if these cells were plated onto a substrate whose stiffness resembled the one found in the brain (0.1–1 kPa), muscle (8–17 kPa) or bone (25–40 kPa), respectively. In addition, the researchers found that this lineage commitment was abrogated by the inhibition of the motor protein non-muscle myosin II (NMM II) by treating cells with the cytoskeletal drug blebbistatin [Within the nucleus, chromatin is organized in nuclear topological domains that differ depending on the cell type and stage of differentiation. The nuclear spatial organization, or nuclear architecture, ensures the correct performance of the transcriptional programs within cells, thus giving it an important role in cellular differentiation and development. Furthermore, nuclear architecture is dictated not only by the epigenome but also by the nuclear lamina, which both physically supports the nucleus and anchors chromatin to the nuclear envelope . This sur lamina . MoreoveΔ cells) . It has Δ cells) . A direc protein . In addibistatin . Thus, iIn this review, we will focus on the current knowledge about the role played by chromatin organization and nuclear lamina in mammals’ neurodevelopment. We will first give an overview of the three-dimensional organization of chromatin and the nuclear envelope structure. Afterwards, we will give a general introduction about the developmental process of the nervous system. We will then discuss examples showing the importance of chromatin organization and nuclear lamina in the development and differentiation of neuronal cells. Drosophila, insulators can interact with each other at high distances, and this interaction can either stabilize or suppress the enhancer–promoter communication depending on the orientation of the enhancer and promoter relative to the insulator elements [The three-dimensional organization of chromatin within the nucleus is critical for the regulation of gene expression and cellular function. In eukaryotic cells, the DNA is organized with different levels of compaction to fit within the micron-sized nuclear space. The DNA wraps around a complex of eight histone proteins constituting the nucleosomes, resulting in the 10 nm fibre known as chromatin that can reorganize into a 30 nm fibre, at least in vitro; however, chromatin is mainly found as a disorganized structure, heterogeneous and diverse in diameter and with a high bendability . Indeed,elements . Howeverelements . This moelements ,32,33. Telements . Other selements . Chromatelements . With reelements . In factelements ,32,37,38elements , with TAelements . Within elements ,41,42  is made up of the outer nuclear membrane (ONM) and the inner nuclear membrane (INM) and acts as a physical barrier that separates the cell nucleus, harbouring chromatin, from the cell cytoplasm . BetweenThe LINC complex (linker of nucleoskeleton and cytoskeleton) spans the INM and ONM and provides mechanical coupling between the actin cytoskeleton and the nucleus ,47. ThisLMNA, LMNB1 and LMNB2. The products of the LMNA gene are lamins A and C, which are translated following an alternative splicing event on exon 10. The LMNB1 and LMNB2 genes encode Lamin B1 and Lamin B2, respectively [In humans, three genes encode the components of the nuclear lamina, ectively . Lamin Bectively ,51,52.The elaborate organization of the nuclear envelope provides structural support to the nucleus and allows the tethering of heterochromatin domains to the nuclear periphery, supporting genome compartmentalization  characterized by a radial alignment and a bipolar morphology. These cells undergo symmetric divisions for self-renewal to increase the size of the cell pool, which, by the end of gastrulation, forms the ral tube . At thisral tube . In the ral tube ,62. InstDrosophila, DamID mapping of LADs showed that differentiated neurons have enhanced, HP1α-rich heterochromatin associated with the nuclear lamina. In contrast, Kc167 cells, of embryonic origin, present LADs lacking HP1α [Epigenetic modifications are usually associated with the regulation of the development and differentiation processes. However, the different compartmentalization of chromatin also plays an important role in these contexts. Studies on murine embryonic stem cells (mESCs) as well as on human embryonic stem cells (hESCs) revealed, through transmission electron microscopy, that undifferentiated ESCs have euchromatin-rich nuclei with prominent nucleoli, whereas differentiating ESCs are characterized by increasingly condensed heterochromatin distributed in a diffuse granular pattern and as a dense strip beneath the nuclear edge . Similaring HP1α . These cNakao’s group also showed that even mESCs are characterized by chromocenters that are smaller than mNPCs and integrated into larger foci in post-mitotic neurons (mPMNs) [. Additionally, the deposition of epigenetic markers is involved, such as the active histone mark trimethylated lysine 4 of histone H3, which increases at chromocenters during neuronal differentiation in the neocortex. This is accompanied by a parallel increase in the transcription of major satellites [Stachowiak’s group identified the TADs in mESCs and mNCCs, showing the relocation of the position of TAD boundaries in mNCCs compared to the mESCs [Drosophila. In vivo cell-type-specific chromatin maps revealed that in neural stem cells (NSCs), the stemness genes, involved in the establishment of NSC identity, and cell cycle genes were found within permissive chromatin marked by H3K27ac, whereas during the differentiation of neurons, they move to a repressed HP1-rich chromatin. In contrast, most pro-neuronal genes are located within TrxG-permissive chromatin in neurons, while in NSCs, they are found in the silent “black” chromatin (lacking epigenetic modifications) or trithorax group (TrxG)-repressed chromatin (containing both H3K27ac and the linker histone H1) [In this regard, the analysis of the three-dimensional organization of the genome during neuronal murine development showed that mESCs are characterized by open chromatin with epigenetic marks of active gene expression , and few compacted chromatin domains. Instead, murine neural progenitor cells (mNPCs) have condensed chromatin with more heterochromatic domains clustered in chromocenters, bright DAPI-positive domains of constitutive heterochromatin ,66,67,68 (mPMNs) ,70,71,72tellites . In accotellites . Intereshe mESCs . Specifihe mESCs , which she mESCs . In addihe mESCs . Finallyhe mESCs . From thtone H1) .LMNA mutation leads to prenatal lethality in humans, whereas in mice, LMNA mutation is not lethal and results in different pathologies a few weeks after birth [Van Steensel’s group analysed mNPC differentiation and discovered an increased interaction between the nuclear lamina and 633 genes, some of which are “stemness” genes, i.e., Nanog, Klf4 and Oct4 [Williams’ group analysed the positioning of Mash1, a proneuronal factor, and noticed that the Mash1 locus is mostly located at the nuclear periphery in ESCs, which, upon neuronal induction, relocates to the interior part of the nucleus. Moreover, this study demonstrates that repositioning is directed in a cell-type-specific manner. Indeed, other differentiated cell types were characterized by Mash1 located at the nuclear periphery, similar to the ESCs [Pcdh9 gene [The redistribution of chromatin is needed during development to specify the cell type’s fate and is essential to cell fitness and function. Alterations in key components of the chromatin 3D reorganization such as the nuclear lamina induce aberrations during development that potentially lead to organism death . Indeed,er birth . Intriguer birth  accordiner birth . Van Steand Oct4 . Since tand Oct4 ,81. Willthe ESCs . Howeverthe ESCs . This isdh9 gene  and seveCarollo and Barra 2023 [Young’s group [Coffinier and colleagues demonstrated that the deficiency of Lamin B1 (Lmnb1Δ/Δ) causes problems in the development of the cerebral cortex in mouse embryos, with impairments in neuronal migration as well [Jung and colleagues demonstrated that the mutated Lamin B1 mislocalises in the nucleus in a honeycomb fashion, which correlates with strong defects in cell nucleus shape. Specifically, it has been observed that during in vitro migration, the NPCs of Lmnb1CS/CS mice have dumbbell-shaped nuclei and blebs. In these cells, Lamin B1 was mainly at the leading edge (towards the direction of the migration). Strikingly, the opposite side of the cell (the trailing edge) was occupied by the bulk of chromatin, called “naked chromatin” because it is disconnected from the nuclear lamina [Considering the strong interdependence between chromatin organization, heterochromatin and nuclear lamina, as discussed above and in rra 2023 , it is n’s group ,84. In 2 as well . This wa as well . In addi as well . Atypicar lamina ,85.LMNA, LMNB1 and LMNB2 and that, more specifically, Lamin B1 depletion causes LAD detachment from nuclear lamina with a subsequent impact on chromatin redistribution and, thus, chromatin dynamics in MDB-MB-231 breast cancer cells [Gigante and colleagues have recently shown that conditional depletion of Lamin B1 in postnatal, quiescent mouse stem cells negatively impacts their commitment towards olfactory sensory neurons, with down-regulation of genes implied in the response to odour stimuli [All the above strongly suggests that Lamin B1 is essential in retaining chromatin bound to the nuclear lamina. Indeed, it has been shown that Lamin B1 is important for TAD–TAD interaction in an mESC model of TKO for er cells ,87. Intuer cells . Moreove stimuli . In addi stimuli ,90.Development is a complex event, with many aspects of cell regulation involved. For instance, changes in the cell microenvironment can result in modifications of the cell’s phenotype, contributing to the determination of cell fate. Nevertheless, the manual of cell differentiation is written on the DNA and, thus, the plasticity of cells depends on its regulation. DNA regulation is, in fact, an intricate and intriguing event in a cell’s life. Epigenetic factors, cis-acting elements (insulators), non-coding RNAs and DNA compaction are important aspects of DNA regulation. The 3D organization of chromatin and the positioning of the genes seem to be involved in the regulation as well, having a role in the determination of gene transcription patterns . Here, wThe focus of this review is neuronal development. However, it will also be of interest to evaluate the changes in the 3D organization of chromatin and how these changes in chromatin arrangement are orchestrated during neuronal maturation, particularly when neurons acquire their specific morphology and, thus, the electrophysiological and molecular characteristics that allow neuronal function in the central nervous system. Additional data would also be needed in order to confirm, in other contexts of cell differentiation, and refine this scenario of chromatin architecture and nuclear lamina collaboration in cell differentiation. This can provide insights into the process of cell differentiation and, more widely, of DNA regulation.


Introduction: In mouse, the zygotic genome activation (ZGA) is coordinated by MERVL elements, a class of LTR retrotransposons. In addition to MERVL, another class of retrotransposons, LINE-1 elements, recently came under the spotlight as key regulators of murine ZGA. In particular, LINE-1 transcripts seem to be required to switch-off the transcriptional program started by MERVL sequences, suggesting an antagonistic interplay between LINE-1 and MERVL pathways.Methods: To better investigate the activities of LINE-1 and MERVL elements at ZGA, we integrated publicly available transcriptomics (RNA-seq), chromatin accessibility (ATAC-seq) and Pol-II binding (Stacc-seq) datasets and characterised the transcriptional and epigenetic dynamics of such elements during murine ZGA.Results: We identified two likely distinct transcriptional activities characterising the murine zygotic genome at ZGA onset. On the one hand, our results confirmed that ZGA minor wave genes are preferentially transcribed from MERVL-rich and gene-dense genomic compartments, such as gene clusters. On the other hand, we identified a set of evolutionary young and likely transcriptionally autonomous LINE-1s located in intergenic and gene-poor regions showing, at the same stage, features such as open chromatin and RNA Pol II binding suggesting them to be, at least, poised for transcription.Discussion: These results suggest that, across evolution, transcription of two different classes of transposable elements, MERVLs and LINE-1s, have likely been confined in genic and intergenic regions respectively in order to maintain and regulate two successive transcriptional programs at ZGA. Upon fertilisation, the fusion of highly differentiated sperm and oocyte cells generates a totipotent embryo. The newly generated embryo is transcriptionally inactive and, in absence of transcription, its development relies on maternally supplied transcripts and proteins originally deposited into the oocyte cytoplasm . HoweverMus musculus (mouse), occur in the early 2-cell and 2-cell stages respectively (Dux (encoded by the Duxf3 gene) which coordinates a specific transcriptional programme characterised by the expression of, among others, the Zscan4, Prame, Eif1a-like gene family members and of the endogenous retroviruses (ERVs) MERVLs  MERVLs . MERVL ec genome .f and Tf subfamilies) act as chromatin remodellers in the 2-cell stage , another class of transposable elements (TEs), play crucial roles during murine ZGA . Evolutill stage . In partll stage . In addi program .Dux/MERVL transcriptional program or LINE-1s are activated by an independent program?In summary, MERVL elements activate a ZGA-specific transcriptional program and LINE-1 RNAs are required to switch it off, allowing the embryo to develop beyond this stage. This model leaves several important questions unanswered. How is the transcription of LINE-1 elements activated at ZGA and from which loci? Is this linked to the In this study, we integrated publicly available RNA-seq, ATAC-seq and Stacc–seq datasets to characterise the dynamics regulating the activation of MERVL and LINE-1 elements at murine ZGA at the transcriptional and epigenetic levels. We identified and characterised a set of LINE-1 elements which result likely transcribed at ZGA minor wave onset, according to our analyses of ATAC-seq and Pol-II binding site datasets. We propose that the transcription of these LINE-1s might be regulated by YY1. Altogether, our results show that MERVL elements are transcribed from gene-dense regions while LINE-1 RNAs are expressed from intergenic regions, suggesting the interplay of two, distinct, transcriptional programs co-ordinately regulating murine ZGA.The RNA-seq data analysed in this study were retrieved from a previous publication . The RNATrimmed RNA-seq reads were mapped to the murine genome (mm10 version—gencode vM22 version) using STAR (v2.6.0c) . DefaultTE locus specific expression levels were calculated using SQuIRE . First, http://broadinstitute.github.io/Picard). Selected reads mapping exclusively on TEs and in the proper orientation were finally counted in each sample.Expression levels of the TE consensus were calculated using TEspeX ; 2022. HDifferentially expressed genes (DEG) between the early 2-cell and the zygote stages were identified using edgeR . NormaliGO enrichment analysis was performed using topGO . GO enrip-values were then calculated and corrected using the FDR Benjamini and Hochberg correction. FDR significant threshold was set to 0.05.Transcription start sites (TSS) of the upregulated genes were defined as the first nucleotide at the 5’ of each given gene. To identify TEs enriched nearby the TSS of the early 2-cell vs. zygote upregulated genes, the TSS of each gene was elongated both at the 5’ and 3’ by 100 nucleotides. The genomic coordinates of the elongated TSS were then overlapped with the genomic coordinates of all the murine TEs using bedtools intersect (v.2.27.0)  and its p-values and FDR were calculated and filtered as already described.Gene clusters in the murine genome were identified using ClusterScan  . Next, tRaw RNA-seq data was retrieved from two previous studies . In the ATAC-seq data were retrieved from a previous publication  accessi. Alpha-an different loci, bowtie2 defines the best alignment  among these n and reports only this to the output file. Next, the Genrich tool was used to perform the ATAC peak calling analysis merging the biological replicates  (https://github.com/jsh58/genrich). Only ATAC peaks longer than 10 nucleotides were retained for further analyses. The same analysis was repeated by using bwa (v 0.7.15-r1140), bowtie1  and bowtie2 to rule out possible biases introduced by the random assignment of non-uniquely mapping reads.Trimmed ATAC-seq reads were mapped to the murine genome (mm10 version—gencode vM22 version) using bowtie2 (v2.3.5.1) . DefaultATAC peaks were annotated respective to the transcript genic features using the R/Bioconductor package ChIPseeker (v1.24.0) . The TxDGenomic coordinates of the LINE-1 monomers were retrieved from a previous publication . MonomerTo define whether the young LINE-1s overlapping early2-cell intergenic ATAC peaks were expressed during the developmental time-course, the expression levels of the LINE-1s of interest were compared to the expression levels of randomly selected young LINE-1s. To this end, the normalised expression levels of the LINE-1s of interest were retrieved from the previous TE single loci expression quantification analysis (see “TE expression quantification—locus specific” section). Expression of contingent LINE-1s whose coordinates were joined (see “LINE-1 monomer annotation”) was summed. The same analysis was then repeated 1,000 times on an equal number of randomly selected young LINE-1s regardless of their ATAC status in the early2-cell stage. This analysis was performed separately for young LINE-1s with  and without  a monomer.Stacc-seq data were retrieved from a previous publication (accession number: PRJNA558961) . In totaMapping to the reference genome, peaks identification and bigwigs files generation were done as previously described for the ATAC-seq data with the exception that no threshold was set on the minimum length of the Pol-II binding sites and that only binding sites scoring an area under the curve (AUC) higher than 500 were selected for further analyses.p-values and FDR were calculated as previously described. The same analysis was also repeated intersecting the end-point coordinate of the same LINE-1 elements. Upset plots were generated by using the ComplexUpset R library (Genomic coordinates of the monomers of the LINE-1s of interest were elongated by 100 nucleotides at both far ends and intersected (bedtools intersect) with the genomic coordinates of the Pol-II binding sites identified in each of the analysed stages. The same analysis was repeated by shuffling 1,000 times the coordinates of the Pol-II binding sites by using bedtools shuffle. Z-scores,  library , ggplot2 library , based o library .runAme function of the memes R package, which is a wrapper of the AME tool of MEME Suite  (Bigwigs track generated in this study for both ATAC-seq and Stacc-seq (Pol-II binding) data are deposited on Zenodo at: ads 250) . In addiads 250)  and the p-values were FDR corrected (Benjamini–Hochberg). *p < 0.05, **p < 0.01, ***p < 0.001.All the statistical analyses performed externally to previously reported software  were conducted either in R (v3.6.2)  or in pyf and Tf, “young L1” refers to the LINE-1s belonging to A, Gf and Tf and “LINE-1” refers to all the LINE-1, with no distinctions among evolutionary ages.In this article, “old L1” refers to all the LINE-1s not belonging to A, Gp < 2.2e-16) with several of the well-known Dux targets belonging to the set of upregulated genes . Gene ontology enrichment analysis revealed that, in agreement with the analysed biological context, the upregulated gene functions were mainly related to GO terms such as transcription regulation, translation initiation, proteolysis and cell death/proliferation (p < 2.2e-16), with the transcription start site (TSS) of 147 of these genes (17%) located in the proximity (<100 nt) of annotated MERVLs, while this is expected by chance for less than 1% of genes in the murine genome  gene analysis between the early 2-cell (ZGA minor wave stage) and the zygote  stages. A total of 1,060 genes resulted differentially expressed between the two conditions with 861 genes being upregulated in early 2-cell compared to zygote  . To define whether this was a feature specific of the minor wave genes or it was characteristic also of other gene sets, we identified the ZGA major wave genes (upregulated genes in 2-cell vs. early 2-cell stage) and overlapped their genomic coordinates with the ones of gene clusters. Of note, cluster enrichment appeared to be ZGA minor wave-specific, as ZGA major wave genes did not display the same enrichment (p = 0.36).Next, we wondered whether other genomic features, besides MERVL enrichment nearby the TSS, characterised the ZGA minor wave genes. In particular, starting from previous evidence showing how high gene/promoter density facilitates the transcription initiation at zebrafish ZGA , we testp < 2.2e-16). In addition, in line with previous evidence (p < 2.2e-16) and B2 elements  (f and Tf subfamilies), ERVs (ERVK and ERV1) and SINE elements (Alu and B4) (t-test p = 1.84E-08)  . The rem and B4) . However and B4) . The maj and B4) . Since i.84E-08) .f and Tf subfamilies) resulted upregulated in early 2-cell compared to zygote . This is in stark contrast with all the other stages where 70% of the peaks resulted annotated in genic regions. A further investigation of the localisation of these 9,553 early 2-cell intergenic peaks showed that more than 50% of them were in correspondence of LINE-1 elements  (p < 2.2e-16). Additionally, such enrichment resulted stage-specific with no other analysed embryonic stage displaying a similar pattern. The results were confirmed either by repeating the analysis of the same dataset with the same tool (bowtie2) or by mapping the reads to the reference genome using other two tools (bwa and bowtie1) to rule out possible biases introduced by the random assignment of non-uniquely mapping reads  . This reng reads . These rng reads . In adding reads  and no eng reads . The enrng reads  as it reng reads . In linef or Tf, 0.2–4 MY) LINE-1 elements and, while the former are usually represented by transcriptionally inactive elements, the latter are more likely to have retained their transcriptional competence (f or Tf), while these subfamilies represent only 4% of the total LINE-1s annotated in the murine genome (p < 2.2e-16). Since most of the LINE-1 elements in the murine genome are 5’ truncated . These results support the idea that the LINE-1s overlapping early 2-cell intergenic ATAC-peaks could be autonomously transcribed. To define the transcriptional dynamics of these LINE-1s, we interrogated again the RNA-seq data. To this end, we compared the expression levels of the 2,116 intact and 1,298 truncated LINE-1s overlapping early 2-cell intergenic ATAC peaks with the ones of randomly selected young LINE-1s. As expected, when lacking an A, Gf or Tf monomer in their internal promoters, both groups of LINE-1s resulted not to be expressed . In addition, Pol-II was enriched also in the monomers of the LINE-1s of interest at the 2-cell stage (p < 2.2e-16). Of note, at the 2-cell stage, Pol-II was enriched also at the 3’ end of such LINE-1s (p < 2.2e-16). Since Pol-II positioning at the transcript 3’ end marks transcription termination (p < 2.2e-16). Analysis of the specific set of LINE-1 monomers bound by Pol-II at the 8-cell stage revealed how 87% of such monomers were bound by Pol-II already at the early or late 2-cell stage  and young  (p < 2.2e-16). We next sought to define the transcriptional profile of Yy1 in the preimplantation embryo. According to the analyzed RNA-seq data, Yy1 mRNA appeared to be part of the maternal transcripts deposited in the oocyte cytoplasm, as its mRNA is already detectable, although at very low levels, in the transcriptionally inactive MII-Oocyte and zygote stages  . In parte stages . In addijor wave . Howeverll stage .The role of MERVL elements as potent regulators of murine ZGA is well known . HoweverThe transcriptional characterisation of the murine ZGA highlights a remarkable number of both genes (861) and TEs  transcriptionally activated at ZGA minor wave. On the one hand, our results confirm previous evidence highlighting that ZGA minor wave genes are enriched in MERVL elements nearby their TSS  and thatloci, reveals that the ATAC-seq peaks are preferentially located at the 3’ end of the LINE-1s of interest. Although the interpretation of open chromatin at the 3’ end of genes/TEs still remains elusive, previous evidence suggests that this phenomenon, at least in early embryos, denotes active transcription  while the maturation happened in later ones. We therefore believe that, at least in this highly dynamic time-course dataset, it is difficult to make direct correlations between ATAC-seq and RNA-seq dataset as the former is probably measuring chromatin accessibility (transcription occurrence/priming) whereas the latter is possibly detecting mature transcripts (transcription termination). Although reasonable, our model is speculative and based exclusively on meta-analysis and therefore needs to be properly validated.This lack of consistency between RNA-seq and ATAC-seq data can be explained in a model in which the protein of a transcriptional activator for these LINE-1s, such as YY1, is not present at the early 2-cell stage. Interestingly, proteomic data  show thaTo further investigate the transcriptional dynamics of the intergenic LINE-1s containing monomers localized within open chromatin at ZGA, we retrieved Pol-II Stacc-seq data identifying Pol-II binding during mouse preimplantation development. Remarkably, Pol-II is significantly enriched in the monomers of the LINE-1s of interest with respect to the rest of the genome as early as at ZGA onset, whereas it does not bind LINE-1s lacking a monomer in their internal promoters. In addition, Pol-II is enriched at both 5’ and 3’ ends of the LINE-1s of interest at the 2-cell stage and at the 5’ end of these LINE-1s at the 8-cell stage. Since Pol-II enrichment at the TTS marks transcriptional termination and that the LINE-1s elements bound by Pol-II at the 8-cell stage were the same bound at early 2- or late 2-cell stage, we hypothesise that LINE-1 transcription started at the early 2-cell stage and terminated at the late 2-cell stage with Pol-II stalling on the monomers of a portion of these LINE-1s, without transcribing them, at the 8-cell stage.in silico prediction, it is interesting to observe how YY1 was previously described to contribute to the silencing of ERVs in mouse embryonic stem cells  in a specific developmental stage (early 2-cell). Even if we could not be sure about the exact location of each specific elements active at a given time, our analysis allowed us to understand that young LINE-1 elements show specific features as a group, which, according to us, paves the way for future and more focused analysis.Dux/MERVL targets whose transcription is started from gene-dense compartments. Altogether, our results suggest that Dux/MERVL and LINE-1 transcriptional pathways are two distinct and antagonistic pathways whose activation at ZGA minor wave is started from spatially separated genomic compartments of the murine genome.In summary, we have identified a set of evolutionary young LINE-1 elements ready to be transcribed at ZGA minor wave onset marked by chromatin accessibility at the 3’ ends and Pol II binding at the promoter regions. In particular, these LINE-1s are confined in intergenic regions, in contrast with


Chromatin architecture, the three-dimensional organization of DNA and its associated proteins, plays a pivotal role in gene regulation and disease. Recent advances in genomics and molecular biology have shed light on the intricate relationship between chromatin architecture and various biological processes. This Research Topic aims to highlight the significance of understanding chromatin architecture in unraveling the complexities of gene regulation and its implications for human health and disease.Kyrchanova et al. review the enhancer-promoter interaction and chromosomal architecture. They discuss the difference of chromosomal architecture between mammals and Drosophila. Scott et al. review how changes in the mechanical environment induce alterations in chromatin architecture. They discuss the critical questions need to be addressed to give insights into the mechanically induced disease.Chromatin architecture influences gene regulation by regulating the accessibility of DNA to transcriptional machinery. It orchestrates the interactions between regulatory elements, such as enhancers, promoters, and insulators, and their target genes. Elucidating the mechanisms by which chromatin architecture modulates gene expression holds the key to understanding fundamental biological processes, including development, cellular differentiation, and environmental responses. Yu et al. examine the 3D genome characteristics at the RT-switching regions. This study expands our understanding of the relationship between RT, histone modification and 3D chromatin structure in cell differentiation.DNA replication is a highly regulated process that ensures the accurate duplication of genome prior to cell division. The replication timing (RT) of different regions of the genome is not uniform and has been found to correlate with chromatin architecture . Yu et aGridina and Fishman review how the organization of the cancer cell genome differs from the healthy genome at various levels. They discuss how these changes in genome organization contribute to cancer development.Aberrations in chromatin architecture have been implicated in various human diseases, including cancer, neurodegenerative disorders, and developmental abnormalities. Dysregulation of gene expression due to altered chromatin structure can disrupt delicate cellular homeostasis, leading to disease initiation and progression. Wang et al. examine the alterations of chromatin organization in doxorubicin-resistant breast cancer and identify potential diagnostic or therapeutic targets associated with the reorganization of chromatin architecture. This study highlights the connection between 3D genome reorganization, chromatin accessibility, and gene transcription and provides valuable insights into the epigenomic mechanisms underlying doxorubicin resistance and offers potential targets for breast cancer treatment.Unraveling the connections between chromatin architecture and disease provides crucial insights for the development of novel diagnostic, prognostic, and therapeutic strategies. The study of chromatin architecture in gene regulation and disease represents a rapidly advancing field that offers unprecedented insights into the fundamental mechanisms governing cellular processes and disease pathology. By understanding the principles underlying the intricate orchestration of chromatin architecture and gene regulation, researchers can pave the way for innovative approaches to diagnose, prevent, and treat a wide range of diseases. Continued exploration of this Research Topic has the potential to redefine our understanding of human health and open new avenues for personalized medicine in the future.