The forkhead box (Fox) transcription factors (TFs) are widespread from yeast to humans. Their mutations and dysregulation have been linked to a broad spectrum of malignant neoplasias. They are known as critical players in DNA repair, metabolism, cell cycle control, differentiation, and aging. Recent studies, especially those from the simple model eukaryotes, revealed unexpected contributions of Fox TFs in chromosome replication and organization. More importantly, besides functioning as a canonical TF in cell signaling cascades and gene expression, Fox TFs can directly participate in DNA replication and determine the global replication timing program in a transcription-independent mechanism. Yeast Fox TFs preferentially recruit the limiting replication factors to a subset of early origins on chromosome arms. Attributed to their dimerization capability and distinct DNA binding modes, Fkh1 and Fkh2 also promote the origin clustering and assemblage of replication elements (replication factories). They can mediate long-range intrachromosomal and interchromosomal interactions and thus regulate the four-dimensional chromosome organization. The novel aspects of Fox TFs reviewed here expand their roles in maintaining genome integrity and coordinating the multiple essential chromosome events. These will inevitably be translated to our knowledge and new treatment strategies of Fox TF-associated human diseases including cancer. Saccharomyces cerevisiae. It flourishes to be one of the largest classes of TFs in human. The Fox family TFs share a conserved common structurally related DNA binding domain (DBD), the forkhead domain. This domain belongs to a much larger superfamily, the winged-helix. The winged-helix/forkhead class of TFs is characterized by a 100-amino-acid monomeric DBD folded into a variant of the helix–turn–helix motif with three α helices and two characteristic large loops or so-called “wings” [The forkhead box (Fox) family of transcription factors (TFs) spans from unicellular eukaryotes to humans, aside from plants . The Fox “wings” .The DNA-binding specificities of different forkhead proteins were intensively examined . The canBesides various sequence specificities, the roles of Fox TFs might be regulated through additional layers such as protein homo- or hetero-oligomerization and distinct DNA binding patterns, which are discussed in DNA replication is a fundamental process essential for all living beings. Each cell needs to accurately duplicate and distribute the whole set of genetic information into two daughter cells. DNA replication is strictly controlled in an orchestrated manner according to different stages of the cell cycle in all eukaryotes. It is crucial for genome stability maintenance, cell proliferation, and cell fate decision. During the past decades, Fox family TFs were demonstrated to play critical roles in regulating DNA replication and cell cycle through both transcription-dependent and transcription-independent mechanisms.Early work on Fox TFs focused on their function as a general transcription factor to control gene expression to affect DNA replication.Saccharomyces cerevisiae, Fkh1 and Fkh2 proteins bind the promoters of the “CLB2 cluster”, which contains 33 genes, including CLB1, CLB2, SWI5, ACE2, CDC5, and CDC20. They act as transcription factors to ensure the cell-cycle regulated expression of these genes, then drive progression through mitosis after binding to the Cdk1 kinase [In More direct evidence has come from the role of Fhl1 in ribonucleotide reductase (RNR) gene expression. Ribonucleotide reductase catalyzes the rate-limiting step in the de novo biogenesis of deoxyribonucleotide triphosphates (dNTPs). It usually comprises a homodimer of large subunit, Rnr1, and a heterodimer of two small subunits, Rnr2 and Rnr4. Another large subunit, Rnr3, is only induced through a multi-level surveillance system when cells suffer replication stress or DNA damage ,7. HeterSimilar to Fox TFs in yeast, the expression of FOXM1B is increased at the G1/S transition in regenerating liver. FOXM1B protein directly binds the CDC25B promoter region and regulates the expression of cell cycle proteins essential for hepatocyte entry into DNA replication and mitosis . FOXM1 cThere is also evidence to show that FOXM1 is regulated by B-Myb, which a key TF in the cell cycle regulation of somatic cells and implicated in different types of human cancer. B-Myb is ubiquitously expressed in various cell types. However, the levels of B-Myb in embryonic stem cells (ESCs), embryonic germ cells, and embryonic carcinoma cells are over 100 times than those in normal proliferating cells. B-Myb ablated ESCs show a significantly decreased expression of FOXM1 and c-Myc. ChIP results reveal a specific enrichment of B-Myb at the FOXM1 locus in the binding site 2 region (BS2) . MoreoveBesides operating as TFs for transcription of the genes involved in DNA synthesis and cell cycle control, Fox family proteins were recently shown to have some direct roles in DNA replication timing. DNA replication consists of three stages, initiation, elongation, and termination. Replication initiation can be further divided into at least two steps ,16. FirsBesides such exquisite step-by-step assembly of the replication machine in each origin, DNA replication is also conducted by a global tempo-spatial program throughout the genome. Unlike bacteria, Eukarya exploit a large number of origins ranging from ~500 in yeast to 50,000 in humans. Interestingly, origins do not fire simultaneously but follow some particular timing program ,34,35,36The timing program is established in the late G1 phase, which is called timing determinant point . Based oFKH1 and FKH2 or their binding sites proximal to Fkh-activated origins results in delayed activation of these origins. As a consequence, other origins, referred to as Fkh-repressed origins, become activated in the absence of FKH1 and FKH2, likely due to reduced competition from Fkh-activated origins for dose-limiting replication initiation factors.Fkh1 is first implicated in modulating the late-origin firing and heterochromatin structure of the mating-type locus in a genetic study in budding yeast . Its exa+). This indicates that the pre-RC assembly is not a prerequisite for Dbf4 recruitment. These findings demonstrated that Dbf4 is barely able to bind origins per se, and Fkh TFs are sufficient to recruit Dbf4 to the FBS+ group of origins.In an independent study, through high-throughput yeast two-hybrid screens, Fang et al. identified two novel Dbf4 interactors: Fkh1 and Fkh2 . ChIP anctf19∆ cells revealed a normal enrichment of Dbf4 at early origins such as ARS606 or ARS607 but a reduced binding of Dfb4 to early origins located in peri-centromeric regions. Collectively, these two independent studies elucidated that Dbf4 is preferentially recruited to early origins through distinct mechanisms in a chromatin context-dependent manner. Very interestingly, Tomoyuki and colleagues found that Dbf4 is recruited to the early origins near centromeres through another mechanism . ThroughThe very C-terminal 50 amino acid of Dbf4 mediates its interaction with Fkh1 and Fkh2. The interaction-defective mutant, dbf4ΔC, phenocopies fkh1Δ alleles in terms of origin firing. More importantly, the direct fusion of the DNA-binding domain  of Fkh1 to Dbf4ΔC fully restores the Fkh-activated origin firing. In control, a fusion of DNA-binding defective forkhead mutant with Dbf4ΔC results in no rescue at all. These findings convincingly demonstrated that DNA-binding activity but not the transcription activation activity of Fkh TFs is necessary for the recruitment of Dbf4. In other words, Fkh TFs determine DNA replication timing through direct physical interaction with DDK and are utterly independent on their transcription roles. Intriguingly, genome-wide replication profiles show that Dbf4 C-terminal fusion with either forkhead or an epitope interferes with the early replication of pericentromeric origins. It was noticed that the addition of a C-terminal tag may specifically abolish the interaction of Dbf4 with Ctf19. In addition to its role as an essential regulatory subunit of DDK, Dbf4 interacts directly with Sld3, which may be attributed to the direct recruitment of these downstream limiting factors. This represents the first clue that Dbf4 may play a direct role in regulating DNA replication in a DDK-independent way. These studies depict how early origins in the different chromosome context compete for DDK, the upstream rate-limiting factor in determining the replication timing program in G1.RIF1 increases the proportion of hyperphosphorylated Mcm4 and partially compensates for the limited DDK activity in a temperature-sensitive (ts) mutant of the catalytic subunit of DDK, cdc7-1, in yeast [cdc7-4 and dbf4-1 mutants. ChIP results confirmed that the Rif1–PP1 interaction is necessary for PP1 enrichment in late origins in both S. cerevisiae and S. pombe. After docking onto the pre-RC through Rif1, PP1 then reverses the MCM phosphorylation carried out by DDK and represses origin firing. Rif1 can also mediate MCM dephosphorylation at replication forks, and the stability of dephosphorylated replisomes strongly depends on Chk1 activity in animals [Contrary to the stimulatory role of Fkh1 and Fkh2 in origin firing, Rif1-mediated PP1 phosphatase inhibits replication initiation through reversing MCM phosphorylation. Rif1 (RAP1-interacting factor) was originally identified as a telomere-binding factor to regulate the telomere length in yeast . The Masin yeast . Rif1 hain yeast . Mutatio animals .Interestingly, MCM phosphorylation is regulated by additional mechanisms . After lBesides MCM, RIF1-PP1 protects the origin-binding ORC1 from premature phosphorylation and consequent degradation by the proteasome in G1 . MeanwhiS. cerevisiae and S. pombe Rif1. When nine putative DDK or CDK sites in Rif1 are changed to alanine, the temperature sensitivity of cdc7-1 is enhanced, while changing them to mimic phosphorylation (aspartic acid) has the opposite effect.Bringing things full circle, PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK . Indeed,In conclusion, MCM loading and pre-RC assembly occur in all origins throughout the genome, which means that all of them are licensed for replication. However, the replication timing and the efficiency of each origin are determined by the phosphorylation of MCM. This critical event is precisely controlled by protein kinase DDK and phosphatase PP1, which are mediated by Fox TFs (or COMA at pericentromeres) and Rif1, respectively a.Besides helping “the early birds catch the worm”, which means that Fox TFs recruit the limiting initiation factors to early origins , there iARS305V-R) loses early-firing in fkh1∆fkh2∆ cells [ARS305V-R regains early replication in the succeeding S phase. Using this Fkh1-induced origin activation system, Zhang et al. recently labeled origin ARS305V-R with tetO/TetR-Tomato [cdc7-4 andcdc45-1 ts mutants as well as in Fkh interaction defective mutant dbf4∆C. Moreover, an MCM4-14D mutant mimicking the phosphorylation status by DDK can bypass the requirement of DDK. Therefore, the origin mobility depends on MCM phosphorylation by Fkh1-mediated DDK and subsequent Cdc45 loading [Fox TFs were first found to be required for the clustering of early origins in G1 by the Aparicio group . They ob2∆ cells . If Fkh1R-Tomato . They ob loading . These sfkh1 with DSM mutated (fkh1-dsm) cannot dimerize, which binds origins in vivo but fails to cluster them. Therefore, Fkh1/2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance. However, such mutations pose a subtle effect on their transcription function, suggesting that the different binding patterns of Fkh determine their distinct roles in transcription and replication/chromatin organization. Interestingly, Fkh1/2 bind strongly at their transcriptional target genes of the CLB2 group. In comparison, their binding to replication origins and recombination enhancer is relatively weak and dynamic [Precise spatial and directional arrangement of Fkh1/2 sites is crucial for the efficient binding of the Fkh1 protein and for early firing of the origins . Both Fk dynamic .Fox TFs are well-known to maintain genome stability through participation in the process of DNA damage response and repair. Here, we summarize their crucial physiological roles in DNA replication, providing a new aspect of this highly conserved family of TFs because they participate in most, if not all, essential biological processes critical for cell fate decisions, including cell cycle progression, cell proliferation, cell renewal, cell differentiation, cell migration, and cell survival. Additionally, the expression of Fox TFs is frequently upregulated in many types of tumors. For example, the upregulation of FOXM1 expression is an early event during cancer initiation, progression, invasion, metastasis, and drug resistance ,68. TherDo FOX TFs determine replication timing in higher eukaryotes? Which FOX TFs are required?In addition to a critical DDK regulator, are there other transcription-independent roles of FOX TFs in DNA replication?Are there more FOX TFs participating in regulating DNA replication and maintaining genomic stability?Do FOX TFs participate in other chromosome processes such chromosome segregation?Are FOX TFs involved in high-order chromosome organization in higher eukaryotes?Despite the rapid-growing knowledge of Fox TFs in the regulation of chromosome replication and structure, there are still some key scientific questions ahead, for example:


Aging represents the multifactorial decline in physiological function of every living organism. Over the past decades, several hallmarks of aging have been defined, including epigenetic deregulation. Indeed, multiple epigenetic events were found altered across different species during aging. Epigenetic changes directly contributing to aging and aging-related diseases include the accumulation of histone variants, changes in chromatin accessibility, loss of histones and heterochromatin, aberrant histone modifications, and deregulated expression/activity of miRNAs. As a consequence, cellular processes are affected, which results in the development or progression of several human pathologies, including cancer, diabetes, osteoporosis, and neurodegenerative disorders. In this review, we focus on epigenetic mechanisms underlying aging-related processes in various species and describe how these deregulations contribute to human diseases. Aging is a multifactorial biological process of declining physiological functions increasing the susceptibility to aging-related chronic diseases, such as cancer, metabolic, cardiovascular, musculoskeletal, as well as neurodegenerative diseases . NumerouEpigenetics refers to reversible heritable mechanisms, which can affect gene expression without underlying changes in DNA sequences, but rather via chromatin modifications. Eukaryotic chromatin is a highly condensed structure containing repeating structural subunits, the nucleosomes. Each nucleosome consists of a histone octamer assembled of two copies of each histone , wrapped around by 147 base pairs of DNA .Epigenetics is a rapidly evolving research field and there is a profound interest in therapies targeting epigenetic as well as aging-related processes. In this review, we focus on aging-associated epigenetic regulatory mechanisms and highlight their implications in aging-related diseases.Saccharomyces cerevisiae, a nucleosome loss of approximately 50% was detected. As a consequence, global transcription levels were highly upregulated in aged cells [The DNA is organized into complex three-dimensional structures; however, for gene transcription, the DNA sequence has to be accessible to key regulators, such as transcription factors and RNA polymerases. Besides chromatin remodeling, which results in the rearrangement of chromatin structures, the global number of histones defines DNA accessibility . In facted cells . Similared cells ,9.Reduced synthesis of histones together with changes in chromatin structure see  leads toBesides the loss of histones, the exchange of canonical histones  with histone variants was observed in aging organisms. These histone variants display distinct primary sequence and properties compared to canonical histones, thereby regulating gene transcription programs. Various aging-related studies evaluating histone variants in murine, primate and human cells implicate a high enrichment of macroH2A (mH2A), H3.3 and H2A.Z. In general, the incorporation of histone variants into the chromatin can be replication-coupled or replication-independent. The replication-coupled process results in a genome-wide incorporation of new nucleosomes into gaps between pre-existing nucleosomes. In contrast, the replication-independent addition of nucleosomes or subunits occurs locally. Thus, during the replication-independent process, histone variants can replace canonical histones, thereby potentially altering gene expression programs ,12. The Caenorhabditis elegans. Here, the deletion of H3.3 resulted in profound transcription changes of longevity-associated genes and in decreased survival [Another example is the H3 variant H3.3, which differs from the canonical form by only four amino acids. It was shown to be incorporated only in a replication-independent manner and to be enriched in transcriptionally active chromatin regions. Recent aging studies in mice revealed that H3.3 accumulates in various tissues during aging and that the canonical isoforms have been almost completely replaced by this histone variant by the age of 18 months . Moreovesurvival .Another well-characterized histone variant is H2A.Z, which only shares approximately 60% similarity with the canonical H2A form. There are numerous studies elucidating the function of H2A.Z; however, some findings are contradictory. For instance, H2A.Z was associated with transcriptional activation, transcriptional repression, cell cycle control, DNA replication and DNA damage repair . A recenA variant of H2AX, which is phosphorylated at the C terminal serine-139 by Ataxia-Telangiectasia-Mutated and Ataxia Telangiectasia and Rad3-related (ATM/ATR), appears during the response to double strand breaks (DSBs). Radiation induced phosphorylation of H2AX in short time ranges, referred to as γH2AX, can be used as biological dosimeter. Together with Senescence-Associated β-galactosidase  staining, γH2AX is frequently used to detect senescent cells, highlighting DSBs and telomere shortening ,22,23.Besides histone methylation, DNA can be directly methylated through the covalent linkage of a methyl group to the fifth position of the cytosine ring to generate 5-methylcytosine (5mC). This modification is mainly present in DNA regions rich in cytosine-phospho-guanine (CpG) dinucleotides. While there is extensive evidence that DNA methylation at promoter regions is associated with gene silencing, the decipherment of the function of gene body methylation is still ongoing ,25,26. TThe conversion of 5mC to the unmodified state is thought to be mediated in an “active”, enzyme-dependent or in a “passive” demethylation process. The family of Ten Eleven Translocation (TET) proteins, TET1, TET2, and TET3, are able to erase DNA methylation in an “active” stepwise process ,32. ThesCaenorhabditis elegans and Drosophila melanogaster [C. elegans, which is thought to be regulated through the DNA demethylase NMAD-1 and the DNA methyltransferase DAMT1 [Drosophila [While methylation-associated control of gene expression pattern is essential for mammalian development and further cellular processes, it was thought to be dispensable in several organisms such as nogaster . Recent se DAMT1 . In addiosophila .While it has been known for several decades that DNA methylation can regulate gene expression patterns, biological consequences are still not fully investigated. Generally, it has been described that CpGs at promoter regions display hypermethylation while other CpGs undergo hypomethylation during aging . RemarkaThe chromatin is organized in a highly compact structure and in order to initiate cellular processes such as transcription, DNA replication, and DNA damage repair, it must be remodeled to enable the accessibility for required factors to the DNA. The reorganization of chromatin structures is facilitated by ATP-dependent chromatin remodeling complexes and results in activation or repression of transcription. These remodelers are multi-subunit complexes containing a highly conserved ATPase subunit which belongs to the superfamily II helicase-related proteins . Based oC. elegans, SWI/SNF and DAF16/FOXO co-localize at DAF16/FOXO target promoters to induce transcription of genes associated with stress resistance and longevity [C. elegans homolog of CHD3/CHD4, increased lifespan and stress resistance and this phenotype depends on DAF16/FOXO activity [Recent data implicate that the two mutually exclusive catalytic ATPase subunits of the SWI/SNF complex, BRM (SMARCA2), or BRG1 (SMARCA4), are involved in telomere maintenance. BRG1 was identified as a negative modulator of the human telomerase reverse transcriptase (hTERT), an enzyme maintaining telomere ends. It was discovered that BRG1 levels are negatively correlated with hTERT in human cervical cancer cells and that BRG1 knockdown promoted hTERT transcription levels . In a laongevity . Mutatioactivity .Similar to SWI/SNF, INO80 is essential for murine telomere maintenance and its deletion resulted in cellular proliferative defects and activation of p21-dependent cellular senescence .S. cerevisiae since its deletion increased yeast replicative lifespan. By de-repressing stress response genes, the deletion of ISW2 contributed to promoting stress resistance pathways [C. elegans provided evidence that isw1 gene expression is upregulated in response to multiple stressors changing histone levels. Accordingly, deletion of ISW1 resulted in reduced lifespan in the P0 and F1 generation, suggesting that ISW1 regulates longevity in worms [ISW2, the catalytic component of the ISW2 complex, was described as a regulator of aging in pathways . Subsequin worms .N-terminal tails protruding from the four core histones can be subjected to post-translational modifications. Depending on the histone, residue and type of modification , these histone modifications can activate or repress transcription . ImportaWhile DNA methylation is highly associated with transcriptional repression, histone methylation can have both activating and repressing effects on gene expression. Previous studies imply that methylation of H3K4, H3K36, and H3K79 promotes transcription and that H3K9, H3K27, and H4K20 negatively affect transcription levels .C. elegans, the trimethylation of lysine 4 of histone H3 (H3K4me3) was demonstrated to negatively affect longevity. In fact, the loss of the H3K4me3 methyltransferase SET2 as well as WDR5 and ASH2 promoted survival, while the depletion of the H3K4 demethylase RBR2 showed the opposite effect [Drosophila where the loss of Lid, the ortholog of RBR2, reduced life span in male flies [In e effect . This file flies . Notablyle flies . In agrele flies .in S. cerevisiae revealed that a deficiency in H3K36 methylation decreased life span. Accordingly, deleting the H3K36me2/3 demethylase RPH1 increased H3K36me3 levels and extended survival in yeast [C. elegans and was associated with a transcriptome-wide age-dependent change of gene expression patterns. When inactivating the methyltransferase MET1 in Drosophila, animals displayed lower H3K36me3 levels as well as reduced lifespan [C. elegans. When deleting the H3K36 dimethyltransferase SET18, lifespan was extended in a DAF16-dependent pattern [A survival screen in yeast . The declifespan . Recentl pattern . DespiteC. elegans. By depleting the demethylase UTX1 and thereby increasing H3K27me3 levels, the lifespan was increased [The trimethylation on lysine 27 of histone H3 (H3K27me3) is mediated by the Polycomb Repressive Complex 2 (PRC2) and removed by Ubiquitously Transcribed TPR on X 1 (UTX1). H3K27me3 negatively affects gene transcription and was shown, together with UTX1, to be reduced during aging in ncreased . Surprisncreased ,63. Morencreased ,64.Drosophila when the deletion of KDM4A, a H3K9me3 demethylase, resulted in impaired wing extension and decreased lifespan in male flies [SUV39H1 was found during aging of human and murine hematopoietic stem cells [The relevance of H3K9me3 was implicated in le flies . Accordile flies . In addiem cells .Besides modifications of histone 3, also the trimethylation of lysine 20 on histone 4 (H4K20me3) has been linked to aging. When kidney and liver samples were collected from rats aged 10, 30, 300, and 450 days, a significant enrichment of H4K20me3 was detected in animals older than 30 days. In contrast, the amounts of mono- and dimethylated H3K20 did not change during aging processes . In addiThe addition of an acetyl group to the ε-amino group onto a lysine residue of a histone is believed to neutralize the positive charge of the lysine. As a result, the interaction between DNA and histone is weakened, thereby loosening chromatin structures and activating transcription. The addition and removal of acetyl groups is orchestrated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively.Sirt6 in mice resulted in severe metabolic effects and an overall survival of only four weeks [Sirt6 deletion resulted in early lethality in mice was supported by another study. Here, it was discovered that inflammatory signaling pathways are targeted by SIRT6. It was shown that SIRT6 represses the expression of Nuclear Factor kappa B (NF-κB) target genes by deacetylating H3K9ac on NF-κB promoters [Sirt6 knockout mice was described to be regulated via the insulin-like growth factor 1 (IGF1) pathway [Sirt6 suppresses senescence as well as apoptosis [When investigating histone modifications in livers isolated from 6, 15, and 30 month old rats, it was discovered that H3K9ac levels decreased with age . In addiur weeks . The obsromoters . In addi pathway . In agrepoptosis .After the detection of H3K56ac in yeast , it was The deacetylase SIRT7 positively regulates HAT1, augmenting the acetylation of H4K12 . The funIn addition, the H4K16 acetylation holds an important role in the regulation of telomere silencing, nucleosome assembly and maintaining chromatin structure ,80. The The histone variant H2AX and its phosphorylation on serine 139 (γH2AX) have been tightly connected to aging since they depict an early event in the DNA damage response. Despite appearing in healthy brain areas, the cerebral cortex of senescent mice displayed a strong abundance of γH2AX at double strand breaks and repair sites . In addiDrosophila, and verified as evolutionarily conserved in humans [Histone ubiquitination constitutes a post-translational modification, the transfer of a small regulatory protein, ubiquitin, to histone core members H2A and H2B. While the H2A ubiquitination mostly inhibits gene expression, H2B ubiquitination enhances transcriptional activity. H2B monoubiquitination is necessary for trimethylation of H3K4 and H3K79 and controls the binding of Cps35 with COMPASS complex, actively regulating transcription ,85,86. Tn humans . In human humans .MicroRNAs (miRNAs), short and noncoding single-stranded RNAs (19-22 nucleotides), have become an emerging aging research interest over the last years. Through sequence-specific binding to their gene targets, miRNAs are able to repress translation or induce mRNA degradation ,90. TherC. elegans. One of the first discoveries in this field was that the miRNA lin-4 targeting the transcription factor lin-14 was not only required during the development, but also for aging processes in C. elegans. Lin-4 overexpression or interfering with lin-14 activity extended lifespan in a DAF16/HSF1-dependent manner [Drosophila where the loss of mir-125 (a homolog of lin-4) reduced lifespan in male flies [The most widely used model organism to evaluate the role of miRNAs in aging is t manner . These fle flies . HoweverWhile there is no to little knowledge regarding miRNA functions in aging across species, a recent study analyzed whole-blood samples from 5000 individuals and identified 127 miRNAs expressed in an age-related pattern (r = 0.70) . In addiEpigenetic discoveries helped to lay the foundation for a deeper perception of multiple diseases. For instance, epigenetic events can contribute to the “hallmarks of cancer” , having led to a revised “hallmark” definition ,98. HereVHL, was associated with angiogenesis, leading to an enhanced supply for the tumor environment. Cell death was shown to be impaired by epigenetic modification of apoptotic or cell cycle key players including CDKN2A, hypermethylation of which leads to a loss-of-function gene in numerous cancers [DNA methylation patterns and miRNAs can influence chromatin state regulation ,100. In  cancers ,100,101.n = 600) in order to identify candidates delaying replicative senescence. Upon treatment with shRNA directed against p300, the replicative lifespan of fibroblasts was substantially increased, accompanied by a reduced occurrence of telomere dysfunction-induced foci (TIFs) [flox/flox) in which the deletion of p300 led to reduced survival after acceleration of leukemogenesis. An enhanced self-renewal of hematopoietic stem/progenitor cells, combined with a decreased apoptosis rate and upregulation of cytokine receptor genes in p300-deficient cells drove leukemogenesis, identifying p300 as tumor suppressor [Distinct chromatin states can favor oncogene activation driving cells to the hallmarks of cancer ,100. Fori (TIFs) . While tppressor . In contppressor . Thus, tSimilarly, a dual function was described for the histone methyltransferase and Polycomb Repressive Complex 2 component EZH2, which has been proposed both as a tumor-suppressor and oncogene . A gain-Leukemogenesis has been found to be linked to further aberrant fusion proteins comprising RUNX1, RARα and CBFB ,116. In DNMT1 expression levels, accompanied by DNA hypermethylation. Interestingly, the Wnt signaling pathway was activated in these samples, indicating a positive regulation effect of Wnt signaling members on DNMT1 expression while Wnt signaling inhibition downregulated DNMT1 [Dnmt null mice. In contrast, Laird et al. reduced the DNMT1 activity genetically and pharmacologically, resulting in a colonic hypomethylation and reduced number of intestinal neoplasia in ApcMin mice [As one of the leading causes of cancer-related deaths worldwide, colorectal cancer can be driven by both genetic and epigenetic alterations within epithelial cells. An overall enhanced DNA methylation pattern has been found not only in inflammatory but also cancerous epithelial tissue ,119. In ed DNMT1 . The stred DNMT1 . Besidesed DNMT1 ,124,125.Min mice ,127. DedColorectal inflammation, as one of the major risk factors of colorectal cancer, was found to be tightly connected to p16INK4a methylation: Wang et al. detected hypermethylation in the promoter region of p16 in human colonoscopic biopsies of rectal inflammatory mucosa, while Hsieh and colleagues suggested the hypermethylation of the p16INK4a promoter region to occur early during the neoplastic progression of ulcerative colitis in colectomy specimens ,124. ConIt is broadly accepted that inflammation is a common event during aging, referred to as “inflamm-aging” . This prSimilarly, NF-κB mediates acute, as well as chronic inflammation, and is proposed as one of the key regulators of aging. Via its transcriptional activity, the NF-κB family induces the expression of cytokines and genes associated with apoptosis and senescence as described elsewhere . NF-κB lAnother epigenetic modification, which has been demonstrated to regulate NF-κB activity during inflammation, is the monoubiquitination at lysine 120 of histone H2B (H2Bub1). This monoubiquitination is performed by the RNF20/RNF40 E3 ligase complex and leads to increased chromatin accessibility. This results in eased passage of RNA Polymerase II and highly active transcriptional elongation . RecentlIn general, the transformation from chronic inflammation to cancer can be promoted via DNA methylation, histone modifications, chromatin remodeling and noncoding RNA regulation, upon which the most important pathways are NF-κB- and STAT3-related ,145. The+ T cell population increases with age, possibly giving rise to associated diseases [Interestingly, the “epigenetic clock” concept of Horvath, has been demonstrated to be very accurate when methylation levels of CpG sites from white blood cells, the central regulators of immune response, were used ,150,151.diseases ,156,157.+ T cells led to the definition of an evolving autoimmune epigenotype [IL10 and IL19 expression. Mechanistically, the reduced phosphorylation of histone 3 serine 10 (H3S10P) and impaired SP1 phosphorylation at the IL10 and IL19 promoter regions impaired IL10 expression. This causes an imbalance towards proinflammatory cytokines (compared to anti-inflammatory IL-10 and IL-19), leading to inflammatory bone loss [Excessive inflammatory response, immunosenescence, and autoimmunity outline the other detrimental side of the inflammatory spectrum. The identification of hypomethylated apoptosis-related genes in naïve CD4genotype ,159,160.one loss . Similarone loss , which wOsteoporosis is tightly linked to aging via epigenetic changes in mesenchymal stem cells (MSCs) . PhysiolRUNX2, the transcriptional activation of the methyltransferase DNMT1 can be inhibited via 5-AzaC treatment. As expected, 5-AzaC leads to a hypomethylation of genomic DNA resulting in increased expression of RUNX2, Osteocalcin (OCN) and Osterix (OSX). The beneficial effect of 5-AzaC was supported by Zhou et al. in cell culture experiments using MSCs. Besides a global reduction in methylation levels, an increase in osteogenic gene expression was detected as demonstrated via enhanced alkaline phosphatase (ALP) activity and, subsequently, aggrandized mineralization [To prevent the hypermethylation of lization . Tet1 and Tet2, factors, which are able to erase DNA methylation. During normal osteogenesis, TET1 and TET2 levels were enhanced with an increased binding to the Osterix promoter [Runx2 expression in Tet1−/−creTet2fl/flPrx1 mice. After the authors demonstrated that the use of siRNAs against TET1 and TET2 led to reduced stem cell properties in bone marrow MSCs (BMMSCs), they found via RNA-seq and qPCR in their mouse model that miRNAs targeting Runx2 gene expression were significantly higher in the knockout mice and a treatment with mimics of these miRNAs increased Runx2 expression in BMMSCs. The authors used chromatin immunoprecipitation (ChIP)-qPCR to demonstrate that TET1 and TET2 directly bind to the CpG island of the P2rX7 promoter, a gene which has been linked to exosome release in earlier studies. After a depletion of Tet1 and Tet2, the subsequent methylation led to impaired self-renewal and differentiation potential on the stem cell level in the bone marrow, thus leading to an osteopenic phenotype [RUNX2 expression, and thereby promoting an osteoporotic phenotype. This TET/P2rX7/RUNX2 cascade may serve as a target for novel therapeutic approaches [Tet2 reduction was detected in the hippocampi of aged mice, and the application of high-titer lentivirus encoding for Tet2 shRNA in young adult mice caused deficits in short-term and long-term learning [It was shown in human bone marrow stromal cells (BMSCs) that in osteoporosis the number of clonogenic BMSCs was reduced, corresponding to decreased levels of promoter ,167. Yanhenotype . Rising proaches ,169. Agalearning .Ezh2 expression in MSCs that the differentiation potential into adipocytes was higher compared to vector control cells, along with reduced Runx2 transcription. Subsequent siRNA-mediated EZH2 depletion led to enhanced RUNX2 expression. Using ChIP-qPCR, the authors demonstrated that enforced EZH2 expression in MSCs resulted in increased H3K27me3 on transcriptional start sites of RUNX2, leading to a suppression of osteogenesis and marking EZH2 as positive regulator of adipogenesis and negative regulator of osteogenesis [Runx2 and Osterix expression and subsequently osteogenic differentiation. Accordingly, the authors suggested the H3K27me3 inhibitor DZNep as a potential therapeutic substance for anti-osteoporotic treatment [Mechanistically, an imbalance between histone modifications of osteogenic and adipogenic genes was proposed as an underlying mechanism of the development of musculoskeletal diseases. For instance, HDAC3 promotes osteogenesis and inhibits lipogenesis, while EZH2 and HDAC6 show opposite effects ,171. In ogenesis ,174,175.reatment .This translation of epigenetic discoveries into the clinic has already been established for several years. In fact, bisphosphonates (increasing MiR191c-5p and miR-497-5p) and monoclonal antibodies, such as denosumab, regulate—among others—DNA methyltransferases, histone acetylases, deacetalyses, and other key factors associated with detrimental epigenetic alterations ,174,175.Neurodegenerative diseases are tightly linked to the process of aging. Twin-studies demonstrated that accelerated epigenetic age, i.e., a higher methylation level, was linked to the development and progression of neurodegenerative diseases ,177. AlzHIC1 (p53-mediated DNA-damage response) and Wnt pathway members (synaptic transmission and plasticity). A protective effect of H4K16ac against neurodegenerative diseases has been suggested and therapeutically explored using a miR-149-5p inhibitor to increase H4K16ac levels in the AD in vitro model 293/APPsw, resulting in reduced β-amyloid formation, potentially attenuating AD progression [Rest in mouse brains (lx/lxNestin-Cre:REST) promoted age-related neurodegeneration, the neuroprotective effect of REST was confirmed by transgenic expression of human REST in C. elegans, reducing the sensitivity to oxidative stress and Aβ toxicity [Rest overexpression suppressed miR-124 while, consequently, the inhibition of miR-124 decreased tau aggregation and rescued memory deficits in 10-month-old P3001S mutant mice [In our aging society, Alzheimer is displays a major health burden and cellular senescence was suggested as a key regulator controlling the shift from physiological aging into neurodegeneration . Interesgression ,181,182.toxicity ,184. Sumtoxicity . In addiant mice . Moreoveant mice ,188.SNCA) gene is crucial. DNMT1 interacts with α-synuclein and is required for its localization in the cytoplasm, thereby reducing its effect on biophysical properties of the DNA [SNCA, which is required for the interaction with DNMT1, may influence the susceptibility to PD [SNCA locus in multiple brain regions associated with PD via human genome-scale enhancer identification via ChIP-seq. A possible targeting approach of this hyperacetylation was found by Mittal et al. who used human SK-N-MC neuroblastoma cells to detect compounds to decrease SNCA expression. Β2AR (β2-adrenoreceptor) agonists were found to modulate SNCA transcription through H3K27 deacetylation at its promoter and enhancer sites, promoting dopamine neuron health by reduced SNCA expression. The authors subsequently demonstrated in a nationwide longitudinal analysis that the most commonly used β2AR agonist, salbutamol, was associated with a reduced incidence rate of PD [tau hyperphosphorylation. SIRT2 inhibition (via AK1), on the other side, improved intracellular trafficking [Drosophila PD models, Sun et al. demonstrated that SMARCA4 was upregulated with aging and a down-regulation of SMARCA4 via siRNAs (drosophila: Brm) in dopaminergic neurons restored life span, indicating possible future diagnostic and therapeutic approaches based on SMRCA4 [In PD, the methylation by DNA methyltransferase 1 (DNMT1) as regulator of the alpha-synuclein  gene, influencing long-term metabolic health even six decades later [One of the earliest indications of a regulatory mechanism beyond sole genetics was deducted from the Dutch Hunger Winter families. In this observational study, an early-life (gestation) adverse environmental factor like famine (in the Netherlands and years of World War II 1944–1945) led to decreased methylation levels at distinct CpG islands, for instance on the Insulin-like Growth Factor II  occupancy at the CDKN2A promoter. In contrast, active histone marks (H3 acetylation and H3K4me2) were detected to a lower extent at CDKN2A promoter region in glucose-restricted medium. Together, glucose deficiency led to a decrease of age-related histone modifications, subsequently, to a reduced incidence of aging-associated diseases [In general, nutritional influences were demonstrated to affect cellular longevity and carcinogenesis by telomerase and CDKN2A (p16) modulation: glucose restriction inhibited cellular senescence via chromatin remodeling and histone acetylation as well as methylation of the  binding ,206. Condiseases ,206,207.On the molecular level, dietary restrictions can be mimicked by polyphenols, a large family of organic compounds naturally contained in fruits, vegetables, and cereals . InteresHIF3A, CPT1A, CD38, and PHGDH were described to be most important [Since aging and obesity have been demonstrated to be closely linked to each other, demographical changes will lead to an increased incidence of obesity in the future . An incrmportant ,215.However, the effects of diet on epigenetic patterns appear to be long-lasting and do not affect just one generation: A high-fat diet in embryonic developmental stages substantially affected later life . The matCHRNA5, GLRA1, and PDX1. Generally, diabetic islets-associated genes showed an overall trend towards increased DNA methylation and a decreased expression of key genes linked with impaired insulin secretion. Pathogenetically, the DNA methylation status  can be directly elevated via high glucose and glycated hemoglobin (HbA1c) while differentially methylated regions in pancreatic islets  promote islet dysfunction [Besides all other aforementioned conditions, diabetes has been previously associated with the involvement of epigenetics. In fact, epigenetic deregulation has frequently been observed in the β-cells of the pancreatic islands which showed an impaired capacity to secrete insulin in diabetes patients . Using pfunction ,221. Anofunction . CDKN1A (p21), PDE7B, and SEPT19 are of predominant interest. It was discovered in pancreatic islands from diabetic and non-diabetic donors that CDKN1A was differentially expressed in diabetic islands. In fact, the impaired glucose-stimulated insulin secretion was associated with an increased expression and reduced DNA methylation pattern of CDKN1A. Via transfection of clonal β-cells with mock-methylated or methylated constructs, the promoter methylation of CDKN1A (p21) has been demonstrated to directly negative affect the transcriptional activity [CDKN2A (p16) regulates the cell-cycle progression to G1 and an overexpression led to decreased cell proliferation, an “aged” phenotype, influencing both islet and non-islet mechanisms driving the diabetic phenotype [The multifactorial and complex polygenicity of diabetes mellitus suggests that the effect size of single genes and CpG sites is low. Among the genes that show decreased DNA methylation and increased gene expression rates in diabetic patients, activity . As a cyhenotype ,224,225.Ezh2fl/fl mice and Cdkn2a−/− mice to reveal that an increased Ink4a and Arf expression in β-cells was linked to a reduced proliferative capacity. While Ezh2 levels declined throughout aging, INK4A levels increased. ChIP analysis uncovered that H3K27me3 occupancy regulating Ink4a and Ezh2 was declining with age, while H3K4me3 and histone acetylation at the Ink4a locus ascended in older mice. The authors concluded from their study that EZH2-dependent histone methylation and repression of the Ink4a/Arf locus are required for β-cell expansion [The majority of association studies has shown multiple gene loci for epigenetic regulation in these central mediators of type II diabetes, β-cells. Chen and colleagues characterized xpansion ,226. In xpansion ,227.In addition, a high relevance of immunoregulatory mechanisms has been detected in type I diabetes. Miao et al. compared blood cells from type I diabetes patients to healthy controls and performed ChIP to identify diabetic methylation profiles. Variations in H3K9ac levels were found at upstream regions of human leukocyte antigen (HLA)-DRB1 and HLA-DQB1 in monocytes from diabetic patients. An increase in H3K9ac levels at the same promoter regions of HLA-DQB1, which was hyperacetylated in type I diabetes patients was found after Interferon gamma (IFNγ) and TNFα stimulation. This suggests that methylation changes in the human leukocyte antigen (HLA) region could precede the development of type I diabetes ,229. HLAUpon further epigenetic regulatory elements in diabetes, micro-RNAs, such as miR-15a and miR-29b, were found to be downregulated in type 2 diabetes, whereas miR-27a and miR-320a were upregulated and might open the possibility for new diagnostic markers ,232,233.This review provides a comprehensive overview of the key role of epigenetic mechanisms in controlling aging as well as the development of aging-associated pathologies. Numerous studies focusing on aging-related molecular mechanisms using cell-based systems, experimental animal models, as well as primary tissues have profoundly contributed to the current knowledge on this topic.It has been shown that distinct epigenetic mechanisms are enriched in aging organisms including the accumulation of histone variants as well as the loss of histones and heterochromatin . In addiWhile the precise mechanisms and connections between several epigenetic changes and human pathologies are still poorly understood, state-of-the-art next generation sequencing methods will allow researchers to address remaining questions. For instance, chromatin accessibility using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) can be coupled to ChIP-seq as well as gene expression studies (mRNA-seq) using bulk mRNA or even analyzing single cells (scRNA-seq). In addition, advances in molecular biology and cell culture approaches /Cas9) will be beneficial in clarifying aging-processes across species. An improved understanding of epigenetic mechanisms affecting longevity will be deciding crucial step towards the identification of new potential therapeutic targets. In fact, epigenetic drugs are of particular interest to the clinic due to their reversible and transient effect.A limitation of manifold epigenetic studies, however, are the variations among single cells , which occur with an even higher frequency in aged organisms ,235. ThiTogether, characterizing molecular changes in different species during aging using state-of-the-art techniques will provide key insights into the relevance of epigenetics of aging and aging-associated diseases.


WNT3A transcription during regeneration of injured cortical neurons. We further demonstrated an increased mono-methylation of histone H3 at lysine 4 (H3K4me1) modification at this enhancer concomitant with a topological interaction between sub-regions of this enhancer and with promoter of WNT3A gene. Together, this study reports a novel mechanism for WNT3A gene transcription and reveals a potential therapeutic intervention for neuronal regeneration.Traumatic brain injury is known to reprogram the epigenome. Chromatin immunoprecipitation-sequencing of histone H3 lysine 27 acetylation (H3K27ac) and tri-methylation of histone H3 at lysine 4 (H3K4me3) marks was performed to address the transcriptional regulation of candidate regeneration-associated genes. In this study, we identify a novel enhancer region for induced  WNT3A gene as a promising RAG of which expression was up-regulated during regeneration of injured cortical neurons }.For histone ChIPs, DIV8-injured or un-injured control cells were cross-linked on DIV9 and DIV10 with 0.8% formaldehyde. Chromatin was sonicated to generate 200-500-bp fragments for subsequent immunoprecipitation of histone-DNA complexes using anti-H3K4me3, anti-H3K4me1, or anti-rabbit IgG antibody-conjugated protein A. DNA was purified in the presence of RNase A and Proteinase K, followed by phenol/chloroform DNA extraction. The immunoprecipitated DNA and input control were analyzed by qPCR, with specific primers for GAPDH. For eRNA quantification, total RNAs were isolated from DIV8-injured or un-injured cortical neurons from DIV8-11 for reverse transcription polymerase chain reaction (RT-PCR). eRNAs of interest were amplified by specific primers and analyzed by DNA gel electrophoresis. Band intensities were quantified by Gel-Pro Analyzer 3.1 and normalized to For 3C template, primary cortical neurons were fixed with 0.8% formaldehyde. Glycine was added to quench the reaction. Cells were centrifuged and the pellet was lysed , incubated on ice for 15 min, followed by centrifugation. NEB 2.1 buffer containing 0.3% of SDS and subsequently 1.6% of Triton X-100 were added to the pellet and incubated at 37 ºC on a shaker. Samples were digested with HindIII overnight at 37 °C. 1.4% of SDS quenched enzymatic digestion at 65 °C for 30 min on a shaker. Ligation using T4 ligase was performed at 4ºC overnight followed by proteinase K and RNase A treatment. DNA was purification and subjected to PCR with specific primers and analyzed by gel electrophoresis.Primers used for 3C:Promoter: 5′-GGGGCCAGGGTCTATAAAAACATGGATG3-3′;e5: 5′-CTGTTGGCACCTTCTTTCTGCTCTCTGG-3′;e7: 5′-GAAAGAGATGAGGGCACAGTGAAGGAGG-3′;LC1 forward: 5′-ACAAGCGAGAGCTAGGACACC-3′;LC1 reverse: 5′-ACTCTTACAAGTTGGCCTTCACTT-3′;LC2 forward: 5′-CCATGGCTTTGATCTTCAAGAACAG-3′;LC2 reverse: 5′-CCAAGGGATAAGTAGCCTGTGTG-3′t-test was performed using Microsoft® Excel® 2016 MSO (16.0.4849.1000) 64 bits. Prism  was used to perform two-way ANOVA followed by Tukey’s test. Statistical significance (*) is defined as P ≤ 0.05.All results are expressed as mean ± SEM from at least three independent experiments unless otherwise noted. Paired Student’s WNT3A gene expression. In response to neuronal injury, H3K4me1-mediated enhancer activation and induced eRNAs transform DNA topology via looping sub-regions of this enhancer to the proximity of promoter to drive WNT3A gene expression during regeneration. We conclude that this novel enhancer−promoter looping mechanism is responsible for the induced WNT3A gene transcription during regeneration of injured cortical neurons.Together, we have identified a novel enhancer for


The eukaryotic genome is hierarchically structured yet highly dynamic. Regulating transcription in this environment demands a high level of coordination to permit many proteins to interact with chromatin fiber at appropriate sites in a timely manner. We describe how recent advances in quantitative imaging techniques overcome caveats of sequencing-based methods (Hi-C and related) by enabling direct visualization of transcription factors and chromatin at high resolution, from single genes to the whole nucleus. We discuss the contribution of fluorescence imaging to deciphering the principles underlying this coordination within the crowded nuclear space in living cells and discuss challenges ahead. A largely unexplored question in chromatin biology is how chromatin organization and dynamics relate to function, a question that needs to be addressed in single living cells and at multiple spatio-temporal scales to be answered. DNA transcription, in particular, is a vital but sensitive process to which many players contribute in order to define and maintain cellular identity . These pChromatin conformation capture methods , 7 are nHere, we describe how recent advances in live-cell imaging of chromatin in eukaryotic cells enriched our understanding of genome organization at different spatial and temporal scales. We start by considering the complementary views that well-established and yet continuously developing sequencing-based “C”-methods (henceforth Hi-C) and imaging-based methods can offer. We then outline various strategies to interrogate chromatin structure and/or dynamics in three-dimensional space and time, from a single gene to the complete genome, and describe how transcription activity influences chromatin folding and dynamics. We finally discuss how physical principles can explain the spatio-temporal coordination between the chromatin structure and dynamics and transcription in the nucleus.Hi-C revolutionized our knowledge of genome structure: a hierarchy of structural elements was discovered, the most prominent ones being DNA loops, topologically associated domains (TADs), and compartments –22. WhilHi-C and derived methods can assess the probability of crosslinking which is a measure of the frequency that chromatin loci are in proximity, probing spatial distances in the range of ~ 400–600 nm . By imagWith the advent of sequential FISH , the thrDespite their success, three-dimensional chromatin structures derived from either sequential DNA FISH imaging or reconstructed from Hi-C maps are currently limited to (i) fixed cells and (ii) a genomic resolution of ~ 2 kb , althougTo gain insights into the dynamic behavior and spatio-temporal organization of chromosomes in live cells, distinct genomic sites can be labeled and tracked in real time Fig. . SeveralFgf5 enhancer and promotor loci in mouse embryonic stem cells (ESC) before and 4 days after inducing cellular differentiation [The prevalence of inactive genes to localize near the nuclear periphery and active genes to prefer the nuclear interior suggests that those loci undergo long-range movements upon gene activation toward the nuclear interior . This cantiation . In contntiation , the stuntiation  reportedntiation . The incntiation . Howeverntiation , 72, stuEarly tools for visualizing chromatin within the entire nucleus either using the expression of fluorescent histones or by incorporating transfected fluorescent nucleotides during replication , 74 contImaging abundant nuclear (macro-) molecules such as chromatin, transcription factors (TF), or RNA poses limitations on the application of common methods such as SPT to analyze their dynamics. Particle identification in a densely labeled environment is not always unique and may even be impossible due to the limited spatial resolution. As a complement to SPT for studying chromatin motion, raster image correlation spectroscopy, pair correlation analysis, and optical flow-based methods have been applied to quantify global chromatin motion in living cells , 78, 79.Transcription is a highly regulated and dynamic process  and is sDrosophila embryos identified three topological states: an open state which is however inactively transcribed and a paired homie–homie state which can be both actively and inactively transcribed [Transcription is not only regulated in time but depends largely also on the spatial organization of TFs and RNAP II . Furthernscribed . Accumulnscribed , 108. ThSeveral mechanisms and models were proposed to explain the nature of the observed chromatin dynamics. Here, we discuss the most popular and experimentally—by fluorescence microscopy and biochemical methods—well-established principles.Upon transcription initiation, hundreds of proteins have to reach a transcription start site in a highly coordinated manner . Live-ceSuper-enhancers, clusters of putative enhancers in close proximity with unordinary high density of transcriptional co-activators binding , 116, haLLPS does not only concern protein condensates such as transcription factories but may also interact with and actively shape the surrounding chromatin environment  , dynamics, and function emerges. However, it must be kept in mind that the nucleus is a technically challenging organelle to analyze. Its content is highly crowded, and there are only few well-structured elements in the nucleus, which would facilitate the description of its interior by serving as reference points. Chromatin is a disordered, extraordinarily long, and confined biopolymer, which is constantly remodeled by a multitude of factors acting on it and that renders the analysis of chromatin and chromatin-associated processes highly complex.Nevertheless, using advanced fluorescence imaging led us to a general picture in which transcription occurs in “transcription factories,” condensates likely formed by liquid-liquid phase separation, to which chromatin is tethered and thus globally and locally constrained. Transcription appears in various aspects to be a key process to take into account in order to understand the dynamic chromatin landscape, even though other factors must also contribute .cis-regulatory elements precede gene activation during Drosophila development and remain regardless of the transcriptional state [cis-regulatory elements. Transient looping events by cohesin and/or TFs, which are likely not to be detectable by ensemble approaches such as Hi-C, could thus constitute a flexible mechanism by which genes can be both activated and repressed, for instance, as a response to stimuli.However, the dependency between chromatin architecture and transcription regulation in vivo is a matter of debate. For instance, contacts between al state . A few sal state , 170. Stal state , and auxal state . Howeveral state –175. GenTo further elucidate how chromatin and transcription are regulated, four main challenges remain in the development of live-cell real-time imaging of nuclear processes and chromatin behavior: (i) sequence-specific labeling, (ii) multicolor imaging, (iii) super-resolution microscopy techniques to allow rapid imaging in three dimensions at a nanoscale resolution of densely distributed emitters, and (iv) improving computational tools that allow processing and visualization of a large pool of information in order to understand stochastic processes.At the single locus level, labeling technologies for tagging of multiple non-repetitive sequences to target specific chromatin domains for live-cell imaging need to be further improved to link chromatin dynamics to function. Applying a lattice light-sheet  illuminaAdditional file 1. Review history.


TF-based biosensors also provide a high-throughput screening method for rapid strain evolution. Furthermore, TFs are important global regulators that control the expression levels of key enzymes in organic acid biosynthesis pathways, therefore determining the outcome of metabolic networks. Here we review recent advances in TF identification, engineering, and applications for metabolic engineering, with an emphasis on metabolite monitoring and high-throughput strain evolution for the organic acid bioproduction.Metabolic regulation of gene expression for the microbial production of fine chemicals, such as organic acids, is an important research topic in post-genomic metabolic engineering. In particular, the ability of transcription factors (TFs) to respond precisely in time and space to various small molecules, signals and stimuli from the internal and external environment is essential for metabolic pathway engineering and strain development. As a key component, TFs are used to construct many biosensors  Saccharomyces cerevisiae (S. cerevisiae)  control the rate of gene transcription by recognizing specific DNA sequences, thus regulating expression of the genome. In addition to the normal biological and physiological roles that TFs play in human cells, they can be used as building blocks and regulatory tools in metabolic engineering and synthetic biology . For exaevisiae) . There aevisiae) . In pastevisiae) , such asevisiae) . When coevisiae) . As an eynthesis . Novel gynthesis . Becauseynthesis . Proteinynthesis , furtherin vivo and performing high-throughput screening for overproducers, which are important methods used to obtain a strain with the desired phenotype from a library of mutants containing a wide variety of genomic alterations.With the rapid development of bioinformatics and genomic editing tools, TFs are playing more important roles in improving the microbial production of valuable chemicals. In this paper, we will briefly review recent advances in the use of microbial TFs to regulate metabolic production of valuable chemical products, with a particular focus on the production of organic acids. We will also summarize strategies for identifying new TFs and review the use of TFs as biosensors for monitoring metabolites Transcription factors are sequence-specific DNA-binding proteins that bind to promoters to either activate or repress transcription . So far,1. Bacterial TF binding sites and related information are also available in some open databases such as CollecTF  and a regulatory domain (RD). Knowledge of binding sites, ligand-protein interaction and binding affinity can help identify an unknown DNA-binding protein. DBDs have been widely characterized both experimentally and bioinformatically, so today, most newly discovered and putative TFs can be identified and grouped by sequence homology to a previously characterized DBD. Now, information on the DBD structures in complex with DNA can be found in the Protein Data BankCollecTF . RD, alsCollecTF . In partin vivo. By binding metabolites of interest, TFs can either activate the expression of reporter genes in a genetic circuit as an activator  to isolate functional biosensors   . Given tSchizochytrium sp. by 57.5% relative to the parent strain after 30 adaption cycles  methods are needed to identify engineered microorganisms that produce the desired products. The application of TF-based biosensors to high-throughput screening has recently been reviewed in detail . Here, wn cycles .E. coli cells was screened by FACS in 30 min -phenylacetylcarbinol  and 2 g/L (K strain)  . More reoduce GA . Raman eiosensor . Recentliosensor , and fouin vivo, historically it has been very challenging to design rational approaches to identifying genes that modulate the production of these compounds. Recently, a FadR-based biosensor was developed to screen for S. cerevisiae genes that increase the fatty acyl-CoA pool using FACS . The combination of genome-wide genomic editing and HTS technologies has drawn increasing attention in the field of strain development. In addition, TFs are also important global regulators that control the expression levels of key enzymes in biosynthesis pathways, therefore determining the direction, flux, balance, and outcome of metabolic networks. Identifying, engineering, and using TFs for applications such as biosensors can help fine-tune gene expression, improve the activity and stability of key enzymes, and direct metabolic flux, thus providing flexible tools for metabolic engineering, as has been demonstrated in the many works reviewed above.In a context of growing concerns regarding climate change, environmental protection, and sustainable development, the biological production of chemicals, pharmaceutical products, fuels, and materials through microbial fermentation of renewable biomass has developed rapidly in the past decade, providing promising solutions for these issues. To make this approach economically feasible in practice, however, the titer, yield, and productivity of organic acid bioproduction, for example, need to be improved further . As a meAlthough there are many examples of successful TF engineering in metabolic engineering and synthetic biology, some challenges remain. To date, the majority of TF-based biosensors have been demonstrated in “proof-of-concept” experiments, with few examples showing real improvement in bioproduction. This is because the type, function, performance, specificity and number of TFs are still limited compared with the large number of host cells, pathways, and metabolites that need to be engineered. The development of novel, high-quality TFs with more functions relies on the further utilization of advanced bioinformatics, computational biology, and protein engineering. In addition, the molecular mechanism, compatibility, robustness, interaction, and quantification of heterogeneous TFs in regulating metabolic networks in host cells need to be understood and thoroughly elucidated to enhance the efficiency of TF-based strain development.J-WL, X-YZ, HW, and Y-PB participated in searching and analyzing literature for this review, as well as writing and critical reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sterol regulatory-element binding proteins (SREBPs) are classical regulators of cellular lipid metabolism in the kidney and other tissues. SREBPs are currently recognized as versatile transcription factors involved in a myriad of cellular processes. Meanwhile, SREBPs have been recognized to mediate lipotoxicity, contributing to the progression of kidney diseases. SREBP1 has been shown to bind to the promoter region of TGFβ, a major pro-fibrotic signaling mechanism in the kidney. Conversely, TGFβ activates SREBP1 transcriptional activity suggesting a positive feedback loop of SREBP1 in TGFβ signaling. Public ChIP-seq data revealed numerous non-lipid transcriptional targets of SREBPs that plausibly play roles in progressive kidney disease and fibrosis. This review provides new insights into SREBP as a mediator of kidney fibrosis via lipid-independent pathways. Numerous studies have revealed the molecular mechanisms of sterol regulatory-element binding proteins (SREBPs) as transcription factors that critically regulate lipid homeostasis . The extCKD, a major public health problem in many countries , is defiVarious factors, such as infiltrating immune cells, albuminuria, and glucosuria in diabetes, have been well recognized to activate proximal tubular epithelial cells, resulting in the secretion of pro-inflammatory and pro-fibrotic mediators that further promote interstitial inflammation and fibrosis development . MeanwhiSREBPs are a family of membrane-bound transcription factors involved in lipid homeostasis. Three isoforms of SREBPs, SREBP1a, -1c, and -2, are encoded by two different genes. SREBP1a and -1c originate from different promotors of sterol regulatory element-binding transcription factor (SREBF)1 genes, whereas SREBP2 is derived from the SREBF2 gene . SREBP1aSREBPs are bHLH-LZ (basic-helix-loop-helix-leucine zipper) transcription factors, synthesized as inactive precursors bound to ER membranes. Each precursor is organized into three domains: (1) an NH2-terminal domain containing the transactivation domain, a region rich in serine and proline, and the bHLH-LZ region for DNA binding and dimerization; (2) two hydrophobic transmembrane segments projected into the ER lumen; and (3) a COOH-terminal domain .2+ metalloprotease, generates transcriptionally active N-terminal domains  in the ER membrane. This SREBP-SCAP complex interacts with insulin-induced gene 1 protein (INSIG1) and INSIG2 . In the  domains , which a domains  , a serine/threonine kinase, is required for the proteolytic maturation of SREBP1c in cultured cells and mice and rat liver. However, the detailed signaling mechanism and its effect on the stability or transcriptional activity of nuclear SREBP1c have not been fully investigated , whereasSREBP1c gene transcription is activated by the liver X receptor (LXR), which is modulated by insulin, PUFAs, and oxysterols. The LXR-retinoid X receptor heterodimer interacts with the LXR-responsive elements located in the SREBP1c promoter, initiating SREBP1c gene transcription . Activat+-dependent deacetylase Sirtuin 1 (SIRT1) deacetylates and inhibits SREBP1c transactivation by enhancing ubiquitination  and its regulatory partner cyclin C (CycC) have been identified to cause the phosphorylation of nuclear SREBP1c at a conserved threonine residue (T402), leading to increased nuclear SREBP1c ubiquitination and degradation. Interestingly, CDK8 and CycC are negatively regulated by feeding and insulin, the most widely studied stimuli that promote SREBP1 activation . ProteinIntra-nuclear SREBPs were initially found to bind to HMG CoA reductase (HMGCR) and LDL receptor (LDLR) gene promoters, known as sterol response elements (SREs). The original SRE sequence is 5′-ATCACCCCAC-3′ . In addiSREBP1 activates lipogenic genes, whereas SREBP2 is more specific for cholesterolgenic genes. SREBP1a and -2 are equally active for SRE, whereas SREBP1c is inactive. However, only SREBP1a and -1c are active for E-box . Due to 1 revealed a higher expression of SREBF1 and SREBF2 in patients with CKD or diabetic kidney disease (DKD) than in normal patients according to the results reported in Ju CKD Glom  gene transcription in fibroblasts. The promoter of collagen (col) VI (α1) contains a growth arrest-responsive region (GARR), comprised of repeat GA-box motifs (GGGGAGGG). In NIH3T3 fibroblasts cultured in serum-free medium, SREBP bound to GARR and induced Col VI (α1) transcription . PlasminAs described in the previous section, the activation of SREBP is classically regulated by cellular sterol levels. Other tissue-injury stimuli, such as mechanical cues  and highGlucose has been shown to activate SREBP1 in renal tubular epithelial cell lines . SeveralParticularly in the kidney, SREBP has then been shown to be activated by several non-sterol stimuli and to contribute to the activation of fibrotic signaling, i.e., TGFβ, a multifunctional cytokine that classically plays a major role in progressive kidney fibrosis .HG increased TGFβ activity via SREBP1 activation in primary rat MCs. As early as 30 min and persisting up to 6 h, 30 mM HG activated SREBPs based on the detection of its mature form (nSREBP1), whereas SREBP2 was not activated. HG promoted SREBP1 binding to the TGFβ promoter, which contains a putative SREBP1 binding site, SRE. This binding was halted by fatostatin and dominant-negative SREBP1a Y335A. Connective tissue growth factor (CTGF) was detected as the downstream target of TGFβ activation. Interestingly, TGFβ promoter analysis revealed that a potential SRE site was within the first 100 base pairs of the start codon. The site is located apparently in close proximity to the Sp1 site, a well-known coactivator of SREBP .Angiotensin (Ang)-II also stimulated SREBP1-induced TGFβ signaling in primary rat MC cultures. Treatment with 100 nM Ang II induced SREBP1 maturation, activated SREBP1 as detected by responsive SRE, and increased the SREBP downstream target, fatty acid synthase (FAS), leading to lipid accumulation. Then, Ang II activated two parallel signals essential for TGFβ promoter activation, ER stress-induced SREBP1 activation and EGFR-mediated activation of co-transcription factor Sp1. ER stress and SREBP1 activation were detected in the glomeruli of Ang II infused mice. SREBP1 inhibition by fatostatin prevented Ang II-induced TGFβ upregulation and ECM accumulation. Mechanistically, Ang II-induced SREBP1 activation in MC culture required angiotensin (AT)1 receptor/PI3K/Akt signaling . PI3K anA recent study suggested the role of lysophosphatidic acid (LPA) in SREBP-induced TGFβ activity . LPA is One should be aware that the cited evidence linking SREBP and TGFβ activation was derived from different experimental conditions or biological contexts. Those studies utilized EMSA and ChiP to discover the binding site motif, but the methods were limited by the skewed distribution of the genomic sequence and the inability to distinguish direct and indirect binding of the transcription factor . A combiActive SREBP1a, but not SREBP1c, activates the TGFβ-responsive reporter plasmid p3TP-lux carrying the promoter region of PAI1 . Due to As discussed above, SREBP1 coordinates TGFβ signaling via its interaction with Smad3 . The actLipid rafts and caveolar endocytosis have been associated with the downregulation of TβRI . CyclodeIn a study by 2. The transcription regulatory peaks were examined from around ± 5 kb of the transcription start sequence of the coding genes -sequencing data, namely ChIP-Atlasng genes . Howeverng genes .As summarized in Claudins are a family of tight-junction membrane proteins that have not been clearly elucidated in the kidney. Genetic mutations in claudin-16 and -19 cause familial hypomagnesemia and hypercalciuria with nephrocalcinosis, whereas polymorphisms in claudin-14 are associated with the risk of kidney stones . In the Syntenin was initially identified as a protein linking syndecan-mediated signaling to the cytoskeleton . SynteniEMILIN is an elastic fiber-associated glycoprotein . In the DAPK is a calcium/calmodulin-regulated serine/threonine kinase that mediates cell death. Deletion of the kinase domain in DAPK attenuates tubular cell apoptosis in renal ischemia-reperfusion injury . DAPK3 iTMEM135 is an LXR-inducible regulator of peroxisome catabolic and anabolic processes mediated via the auxiliary matrix protein import pathway. It also regulates mitochondrial dynamics to protect the retina against oxidative stress and progressive retinal aging . OverexpGiven the importance of SREBP in mediating lipid biosynthesis, which converges with various pathological signaling mechanisms, targeting SREBP is an important pharmacological strategy to attenuate the progression of kidney diseases. The available small-molecule inhibitors of SREBP activation have been comprehensively reviewed elsewhere . Howeverin vitro, and in vivo studies have demonstrated the versatility of SREBPs in mediating diverse biological processes. Particularly in the kidney, SREBP1 acts as an activator of pro-fibrotic signaling by binding to the promoter area of fibrosis-related genes, i.e., TGFβ. The precise elucidation of non-lipid and direct or indirect targets of SREBPs that mediate the development of fibrosis remains a challenge. Emerging data suggest that continued investigation of the SREBP pathway and the discovery of its small molecule inhibitors will facilitate the amelioration of kidney disease via lipid-dependent and -independent pathways (Genome-wide analysis, pathways .DD conceived and wrote the manuscript, and designed the figures. DK and HH provided critical revisions of the manuscript. HH made the final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FOXC1 lead to inherited Axenfeld–Rieger syndrome. Here, we find that FOXC1 and paired box 6 (PAX6) are co-expressed in the human limbus and central corneal epithelium. Deficiency of FOXC1 and alternation in epithelial features occur in patients with corneal ulcers. FOXC1 governs the fate of the corneal epithelium by directly binding to lineage-specific open promoters or enhancers marked by H3K4me2. FOXC1 depletion not only activates the keratinization pathway and reprograms corneal epithelial cells into skin-like epithelial cells, but also disrupts the collagen metabolic process and interferon signaling pathways. Loss of interferon regulatory factor 1 and PAX6 induced by FOXC1 dysfunction is linked to the corneal ulcer. Collectively, our results reveal a FOXC1-mediated regulatory network responsible for corneal epithelial homeostasis and provide a potential therapeutic target for corneal ulcer.Forkhead box C1 (FOXC1) is required for neural crest and ocular development, and mutations in  The outermost surface of the cornea is covered with a non-keratinized stratified squamous epithelium consisting of multilayer corneal epithelial cells (CECs) on the avascular stroma. The self-renewal and regeneration of the corneal epithelium are sustained by a population of limbal stem cells (LSCs), which are segregated in the basal layer of the limbal zone of the peripheral cornea.9 Loss of PAX6 induces the corneal epithelium to acquire an opaque skin-epithelium phenotype, which leads to visual disorder.8Cell fate determination and identity are governed by lineage-specific transcription factors (TFs) and epigenetic modifications. Key TFs not only determine organ development, but also define tissue specificity during adulthood. The master regulator paired box 6 (PAX6) plays a vital role in the developing visual system, and is required for corneal epithelial identity and homeostasis.10FOXC1 mutations in human patients cause ocular anterior segment defects, and pathological corneal neovascularization can be observed in patients with Axenfeld–Rieger syndrome harboring FOXC1 mutations or copy-number alterations.11 Previous findings indicated that Foxc1 maintains corneal transparency and avascularity in the neural crest-derived corneal stromal and endothelial cells.11 Neural crest-specific Foxc1 null mutation in mice can result in disorganized corneal stroma with excessive growth of blood vessels and failure of corneal endothelial formation.11 However, the role of FOXC1 in the corneal epithelial homeostasis and pathogenesis has not been reported.Forkhead Box C1 (FOXC1) is a key TF of ocular development.cis-regulatory elements that are marked by histone H3 lysine 4 dimethylation (H3K4me2). FOXC1-depleted LSCs show the activation of an epidermis-specific gene program and the repression of IRF1 and PAX6, which are pathological characteristics of the human corneal ulcer. These findings indicate that the FOXC1 regulatory network is required for corneal epithelial homeostasis, and FOXC1 may serve as a potential target for corneal ulcer treatment.In the present study, we identify FOXC1 as a novel core regulator that underlies the lineage commitment of human corneal epithelium. FOXC1 regulates the corneal epithelial lineage-specific gene expression and interferon signaling pathways by occupying open To determine whether FOXC1 functions in the corneal epithelium, we first detected its expression pattern during corneal development. We found that Foxc1 was evidently expressed in the ocular surface structure and neural crest-derived cells during the early developmental stage in mice Fig. . The corAs expected, FOXC1 and PAX6 co-expressed robustly in the human corneal epithelium of 13-week and 22-week-old embryos Fig. . Their eTo study the potential relationship between FOXC1 and corneal pathogenesis, we examined 21 biopsy samples from patients with corneal ulcer. Remarkably, partial regions of the diseased corneal tissues from nine patients showed conspicuous absence of FOXC1, PAX6, and the corneal epithelial markers (KRT3 and KRT12). Instead, the keratinized epithelium-specific KRT1 or KRT10 appeared in these lesion areas   in the dCESs indicated their terminal differentiation  Fig. . Expression Fig. . In paraion Fig. .Fig. 3GeKI67 .25 These high-throughput sequencing data showed a high Pearson’s correlation coefficient between two biological replicates . ChIP-Seq data of H3K4me2, which is associated with both the transcription start sites (TSSs) and enhancers,cis-regulatory elements. Interestingly, FOXC1 bound to its own promoter and multiple neighboring enhancers that were marked by H3K4me2 and ATAC  of FOXC1 peaks was located at proximal promoters. In contrast, ~42% of H3K4me2 peaks were located at promoters and ~50% marked putative noncoding enhancers Fig. . ATAC anTAC Fig. , formingTAC Fig. .Fig. 4Ch26 to search TF binding motifs in the FOXC1 and ATAC peaks. We found that both ATAC and FOXC1-bound regions were significantly coenriched for the motifs of key corneal epithelial regulators -mediated knockdown experiments. ZNF750 is known to initiate early- and late-differentiation programs in skin epithelial cells.SCs Fig. . These rFOXC1-deficient LSCs were induced to differentiate into stratified epithelium using the 3D air-lifting culture system. Although epithelial stratification was not affected when FOXC1 was knocked down, the morphology and arrangement of the dCESs were disordered  in dCESs. A total of 2180 genes were regulated by FOXC1 only during CEC differentiation  of the differentially expressed genes induced by XC1 Fig. . For exaT7B Fig. . FOXC1 aZNF750, TINCR, CALML5, SFN, IRF6, KRT1, and KRT108 when FOXC1 was knocked down  and genes response to interferon signaling were significantly repressed in FOXC1-compromised dCESs  activity, and protein kinase B signaling Fig. . Importass Figs. a and 3e.ESs Fig. . FOXC1 rESs Fig. .Fig. 6FOIRF1 expression was also inhibited in LSCs when FOXC1 was knocked down  downregulated TFs with high expression levels, and showed that interferon-induced IRF1 had the highest functional similarity with others Fig. , suggest38 Previous reports demonstrated that FOXC1 is required for ocular anterior segment development in mice and humans, and functional studies on this gene mainly focused on its antiangiogenic role in corneal stroma.40 Here, we identified FOXC1 as a core TF that maintains the corneal epithelial identity during adulthood, which suggested a broader role of FOXC1 in the ocular system.Human corneal epithelium, as a barrier tissue of the eye, suffers from external insults, thus requiring continuous self-renewal and differentiation of resident adult stem cells. LSCs are essential for the maintenance of corneal epithelial homeostasis and visual acuity.FOXC1-depleted dCESs, which indicated that these signal pathways may be important for the corneal epithelial identity. In addition, FOXC1 also controlled protein glycosylation and collagen metabolism in dCESs. Previous findings indicated that FOXC1 in neural crest-derived corneal stroma cells maintains corneal transparency and avascularity by regulating proangiogenic matrix metalloproteinases and VEGF signaling.11 Thus, FOXC1 appears to govern distinct biological processes in different tissues.Specifically, several signaling pathways  were affected in 42 Collectively, our present work provides novel insights into the FOXC1-mediated regulatory network and epigenetic features required for corneal epithelial homeostasis.The present study revealed the chromatin binding profile of FOXC1 in LSCs. The function of TFs in the nucleus depends on the active chromatin environment. Thus, we also mapped the chromatin accessibility and H3K4me2 landscapes. As expected, FOXC1 controlled the corneal epithelial gene network by directly binding to promoters or enhancers marked by ATAC and H3K4me2 signals. Notably, motifs of several regulators important to corneal epithelial self-renewal and differentiation were co-enriched at the centers of ATAC and FOXC1 peaks. The proximity of these TFs implied the potential cooperation between them. This observation supported the notion that cooperative binding of cell-type-specific TFs orchestrates lineage identity and homeostasis.43 Thus, we speculated that FOXC1 could inhibit neo-angiogenesis by regulating interferon signaling in the limbus and corneal epithelium. The anti-angiogenic function of FOXC1 may also be achieved in corneal stromal cells via interferon signaling. Previous report has revealed that the interferon response gene IRF1 is important for the corneal innate immune response to bacterial infection.37 Of note, the expression patterns of FOXC1, PAX6, and IRF1 were consistent in corneal epithelial lineage and pathological cornea. Loss of FOXC1, IRF1, and PAX6 was observed in some regions of the corneal ulcer tissues. These findings raised the possibility that disturbance of the FOXC1/interferon signaling axis causes disorder of the corneal innate immune defense, which results in pathological changes in the corneal epithelium. The role and regulatory mechanism of IRF1 in corneal epithelium fate and homeostasis need to be explored in the future.Interestingly, we found that FOXC1 activated gene expression of the whole interferon signaling pathways in dCESs. Interferon signaling robustly represses angiogenesis in human corneal stroma and tumors.Human embryonic cornea was obtained voluntarily from the donor in compliance with the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (2018006). All human pathological tissues were obtained with the approval of the Ethics Committee of Zhongshan Ophthalmic Center of Sun Yat-sen University (2020KYPZ115).8Human limbus tissues isolated from donors were cut into small pieces and digested at 37 °C with 0.2% collagenase IV (Gibco) for 2 h and then with 0.25% trypsin-EDTA (Gibco) for 15 min. LSC expansion was achieved in Matrigel-coated dishes. The components of the LSC medium have been described previously.LSCs were plated in transwell culture inserts (Corning), and the medium was added to the upper and lower compartments. The cells were cultured for approximately 3–6 days until they reached confluency, after which the medium was removed from the inserts. LSCs were induced to differentiate for another 5–14 days to form dCESs through contact with the medium in the lower chamber. The medium used for air-lifting culture was the same as that used to culture LSCs.8 Briefly, after fixation, dehydration, paraffin embedding, and de-paraffinization, the tissue sections were permeated and blocked with 0.3% Triton X-100 and 3% bovine serum albumin for 1 h. Then, the samples were incubated with primary antibodies overnight and with secondary antibodies for 1 h on the following day. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The following antibodies were used: anti-FOXC1 , anti-K19 , anti-p63 , anti-Ki67 , anti-Cytokeratin 1 , anti-Cytokeratin 10 , anti-PAX6 , anti-Cytokeratin 12 , anti-Cytokeratin 3 , and anti-ZNF750 .Immunofluorescence experiments were performed as previously described.FOXC1 were respectively cloned into the PLKO.1 lentivirus plasmid. LSCs were infected with lentivirus particles encoding shRNAs for 16 h and then selected with 2 μg/mL puromycin (Gibco) for 48 h. The RNeasy Mini Kit (Qiagen) was used to isolate total RNA from cells, and cDNAs were synthesized using the PrimeScript™ RT Master Mix Kit (Takara). qPCR was performed using the iTaq™ Universal SYBR® Green Supermix Kit (Bio–Rad). The following shRNAs were used:Two shRNAs targeting shFOXC1-1: 5′-GAGCTTTCGTCTACGACTGTA-3′shFOXC1-2: 5′-GTCACAGAGGATCGGCTTGAA-3′shPAX6-1: 5′-CGTCCATCTTTGCTTGGGAAA-3′shPAX6-2: 5′-AGTTTGAGAGAACCCATTATC-3′Scramble: 5′-CAACAAGATGAAGAGCACCAA-3′Chromatin was fixed with 1% formaldehyde for 10 min and sheared in a sonification buffer . Fragmented DNA was incubated with primary antibodies overnight at 4 °C and then with Protein A/G Dynabeads  for 1 h. The beads were collected and washed sequentially with the following solutions: high-salt buffer , low-salt wash buffer , and TE buffer .Elution of the immunoprecipitated protein/DNA complexes was performed using elution buffer  at 65 °C for 4 h. Next, the protein/DNA complexes were incubated with proteinase K (Invitrogen) and RNase A (Invitrogen) for 1 h. De-crosslinked DNA was purified using the PCR Purification Kit (Qiagen), and then, the KAPA Hyper Prep Kit  was used to construct DNA libraries for Illumina PE150 sequencing.2, 0.5% IGEPAL CA-630, and 0.1% Tween-20) for 5 min. Then, Tn5 transposase reactions  were performed using the TruePrep DNA Library Prep Kit (Vazyme Biotech). After purification, the transposed DNA fragments were amplified and collected according to manufacturer’s instructions. Library DNA was sequenced with an Illumina HiSeq X10 instrument.In total, 50,000 cells were digested, collected, and lysed in ice-cold lysis buffer . For ChIP-seq data, we used MACS249 to call peaks with the following parameters: -f BAMPE -B –SPMR -q 0.001–call-summits–fix-bimodal–seed 11521 and–extsize 200. For ATAC peak calling, the following parameters were used: -f BAMPE -B –SPMR -q 0.001–call-summits–seed 11521–nomodel–shift -100–extsize 200. The HOMER mergePeaks command was used to obtain overlapping peaks between two biological replicates. Peak distributions were examined with the ChIPseeker package.50 Peak annotations were performed using the HOMER’s annotatePeaks.pl program with the default parameters. Peak summits were used to identify motifs using HOMER’s findMotifsGenome.pl program with the parameter: -size 200.BWA software22 Functional similarity of genes was defined as the geometric mean of their semantic similarities in molecular function and cellular component of GO analysis.Bioconductor R package GOSemSim was used to compute GO semantic similarity between genes according to the published method.supplementary data