Architectural proteins are essential epigenetic regulators that play a critical role in organizing chromatin and controlling gene expression. CTCF (CCCTC-binding factor) is a key architectural protein responsible for maintaining the intricate 3D structure of chromatin. Because of its multivalent properties and plasticity to bind various sequences, CTCF is similar to a Swiss knife for genome organization. Despite the importance of this protein, its mechanisms of action are not fully elucidated. It has been hypothesized that its versatility is achieved through interaction with multiple partners, forming a complex network that regulates chromatin folding within the nucleus. In this review, we delve into CTCF’s interactions with other molecules involved in epigenetic processes, particularly histone and DNA demethylases, as well as several long non-coding RNAs (lncRNAs) that are able to recruit CTCF. Our review highlights the importance of CTCF partners to shed light on chromatin regulation and pave the way for future exploration of the mechanisms that enable the finely-tuned role of CTCF as a master regulator of chromatin. Chromatin, a macromolecular complex of DNA, RNA, and proteins, provides a framework for the packaging of genetic material within the cell nucleus. Its organization plays a crucial role in gene expression and is regulated by a diverse array of protein complexes in response to a dynamic code of histone posttranslational modifications and DNA modifications . CTCF  have been described as crucial factors in chromatin architecture . Recent Originally, CTCF was described in chickens as a protein that binds to a region upstream of the c-myc promoter. Because that binding site has three regularly spaced repetitions of the sequence CCCTC, the protein was named CCCTC-binding factor or CTCF . Later, CTCF has tens of thousands of genomic binding sites, some of which are conserved between species and tissues . CTCF acThe mechanisms underlying CTCF functions are not yet fully understood, but it is probable that most of them depend on interactions with other proteins. One of the better-characterized CTCF protein–protein interactions is cohesin retention. The cohesin ring is a multi-protein complex involved in the formation of chromatin loops . The mecCTCF has a paralogous gene called CTCF-Like (CTCFL), which encodes the protein Brother of the Regulator of Imprinted Sites (BORIS). It is believed that CTCFL originated from a duplication event at some point before the evolution of mammals [Similarly,  mammals . Unlike  mammals . Nonethe mammals ,52.CTCF and BORIS share 75% of identity, mainly in their DNA binding domains, suggesting that they might compete for similar binding sites in the genome. Indeed, BORIS has been described to bind to a large subset of CTCF binding sites; however, there are a few differences in the target regions of both proteins . While CBesides the impairment of chromatin loops, the differences between CTCF and BORIS terminal domains may affect which proteins are recruited upon binding b. ThrougThe chromatin status is dynamic and can be regulated by covalent modification of the amino-terminal ends of histones that protrude from the nucleosome and are accessible to enzymes that chemically modify them through a system of writing, reading, and erasing complexes . These mAmong these interactions, many of the proteins participate in the shaping of the 3D conformation of the genome such as the DNA helicases CHD7 , CHD8 6 and CHD1It is also worth noticing that CTCF interacts with several members of the Polycomb group (PcG). These proteins are part of a system that regulates post-translational modifiers of histones, and their action is generally associated with the transcriptional repression of tissue-specific genes. This group has two members, the Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2). PRC2 is the complex that acts as a writer, as it is responsible for mono-, di-, and trimethylated lysine 27 of histone 3 (H3K27me3). This mark is associated with silenced gene promoters and facultative heterochromatin. H3K27me3 is recognized by PRC1 (reader) that binds to chromatin, monoubiquitinates lysine 119 of histone H2A (H2AK119ub), and prevents transcription by blocking the recruitment of RNA polymerase II ,81. CTCFBesides PcG proteins and chromatin remodeling factors, CTCF’s relationship with proteins related to histone post-translational modifications are remarkable as well. CTCF interacts with proteins involved in the three stages of histone posttranslational modifications . However, we would like to discuss further two particular cases that have not been broadly explored yet; histone and DNA demethylases.Histone methylation is a post-translational modification related to multiple biological functions. Methylation happens mainly in arginine (R) and lysine (K) residues. Arginines can be mono- or dimethylated, and this chemical modification generally potentiates the interaction with other enzymes that modify histone tails . MoreoveCurrently, histone lysine demethylases (KDMs) are classified into two families based on their chemical mechanism of action: the amine oxidase-like and the oxygenase enzymes . The amiubstrate . This faubstrate .In vitro studies have demonstrated that the simple binding of these enzymes to their substrates is sufficient for the demethylation reaction, suggesting that their recruitment must be tightly controlled in order to prevent aberrant demethylation ,93,94. IACTG2, APOE, CTGF, FN1, and TGFβ2, among others. The perturbation of these transcriptional changes could promote breast cancer progression [Until now, few studies have demonstrated the association between CTCF and histone demethylases; in fact, only two KDM partners have been found. The first was reported in 2014 by Yamamoto et al., who found via co-immunoprecipitation that CTCF formed a complex with the H3K4me3 and H3K4me2 specific demethylase KDM5B. Moreover, when conducting ChIP-seq assays, they discovered that KDM5B sites overlap with those of CTCF in most mammary cancer cell lines, and this overlapping phenomenon correlates with a lower H3K4me3 signal compared to those non-overlapping sites a. The rogression ,101.CHD5, the promoter of WRAP53, and the region located at −1922 bp of the ASCL2 transcription starting site. The coexistence of CTCF and KDM4A correlates with the reduction of H3K36me3/2 histone modifications at the first intron of CHD5 and is associated with its transcriptional down-regulation . DNA methylation has several functions; although it is generally associated with transcriptional repression; it is also involved in other vital processes, such as genomic imprinting, X chromosome inactivation, and retrotransposon element suppression ,104. SimThe first evidence of the enzyme-mediated DNA demethylation was observed in 2007, with the identification of the Trypanosoma cruzi enzymes JBP1 and JBP2 that are responsible for gene silencing through the hydroxylation and glycosylation of a thymine methyl group (known as J Base). This discovery pointed toward the existence of “eraser” proteins that are in charge of removing DNA methylation . ShortlyBecause DNA methylation is an epigenetic marker that is essential for correct cellular function and organism development ,112, TETSince TET enzymes form protein complexes with other epigenetic components to modify gene transcription, the interaction of these proteins with CTCF is of particular interest. It is known that synchronized fluctuations of DNA methylation, demethylation, nucleosome positioning, and CTCF chromatin binding have an important role in establishing cell-type-specific chromatin states during differentiation. Loss of CTCF in regions such as the boundaries of chromatin loops, promoters, and TADs can be associated with the spread of DNA methylation and demethylation, and can be linked to the down-regulation of adjacent genes. A hierarchical interaction between cytosine modifications, nucleosome positioning, and DNA sequences controls CTCF binding and regulates gene expression ,126.It has been proposed that CTCF binding to low methylated regions could mediate local DNA demethylation through TET recruitment . The firTaken together, the above information suggests that CTCF could interact, directly or indirectly, with histone and DNA demethylases; it is still unknown whether these complexes are related to repression, activation, or other transcriptional processes.Long non-coding RNAs (lncRNAs) have emerged as important regulators of chromatin structure and gene expression. They act as scaffolds, guides, or decoys that recruit chromatin modifiers to specific genomic regions, mediate higher-order chromatin organization, and influence gene expression . LncRNAsXist promoter, allowing its expression and subsequent X chromosome inactivation [LncRNAs contribute to the functions of CTCF by recruiting it to specific genomic sites, modulating chromatin loops, and regulating the formation of TADs. One of the most studied cases is CTCF-mediated Xist transcriptional repression. Xist is a lncRNA involved in X chromosome inactivation. CTCF represses Xist expression by binding to its promoter; however, Jpx is a lncRNA that binds to CTCF and removes it from the tivation ,134. Rectivation . The inttivation . It has tivation .Several other lncRNAs have been identified to interact with CTCF and modulate its function. HOTTIP, for instance, can recruit CTCF to specific genomic regions and promote TAD formation . SimilarOverall, lncRNAs represent an exciting new area of research in the field of chromatin biology and gene regulation. The interaction between lncRNAs and CTCF offers a new level of complexity to the already intricate network of molecular interactions that govern gene expression and chromatin architecture.CTCF is a nuclear factor that is involved in several chromatin-related processes, including transcriptional regulation, three-dimensional chromatin topology, and epigenetics. Part of its relevance lies in its versatility, as shown in this review, CTCF relies on a broad network of protein and RNA partners to achieve its different tasks. The role of the protein partners is clear upon comparison with CTCF–s and BORIS. In the first case, the lack of the N-terminal domain leads to the loss of the most studied CTCF interacting partners, the cohesin complex. The second case is more complex, since BORIS binding to the DNA has completely opposite consequences than CTCF, besides sharing a high degree of identity at their DNA binding domains. Most of this could be explained by their interactions with different protein partners through their unconserved terminal domains. There is current research going on in this regard, and without a doubt, the study of the interplay between CTCF and BORIS in cancer will help to understand CTCF’s role in chromatin organization and other epigenetic processes.Chromatin is finely organized inside the nucleus through a complex system that has not yet been elucidated. As mentioned previously in this review, many of the epigenetic factors that help to establish and maintain chromatin structure lack DNA binding domains; thus, it has been hypothesized that their action should rely on other proteins. CTCF is capable of binding to thousands of sites in the genome, and due to the flexibility of its DNA binding domain, it is considered a multivalent protein. In this review, we demonstrate that CTCF interacts with a wide array of epigenetic factors which suggests that it could serve as a scaffold for the assembly of different protein complexes. Nevertheless, the logistics involved in partner election, the impact of each complex, and the crosstalk between different partners is an exciting point of view that is worth further study.Since epigenetic markers such as histone and DNA methylation are highly dependent on the chromatin context, the interaction between CTCF and different components of the epigenetic complexes is interesting. So far only a few protein–protein interactions between CTCF and other epigenetic factors have been fully characterized, and in most cases, the studies have been conducted on a specific gene or promoter; thus, genomic scale experiments could be helpful to identify the overall impact and localization of the complexes.LncRNAs add another layer of complexity to the CTCF-mediated chromatin regulation. Currently, the role of most of these complexes in biological processes remains unknown. However, several studies hint towards the existence of a broad CTCF-RNA interaction network. The role of some of these complexes has been discussed here; among them, Jpx is the most remarkable example due to its ability to detach CTCF from its binding sites. Further studies will help to understand the role of CTCF-lncRNA interactions.Because of CTCF’s versatility, it could likely function as a scaffold for many of the epigenetic complexes required for a proper genomic organization. Without a doubt, there are still undiscovered mechanisms for CTCF; the study of this protein could aid to understand the complex mechanisms that regulate chromatin organization and gene expression.


The Research Topic (RT) discussed herein is the second volume of a Research Topic focused on transcription factors (TF) and transcription regulation in the healthy and diseased brain. The previously published first volume was compiled into a well-visited and well-read E-Book  , which is an elementary but complex and dynamic behavior. First, the authors present generalities of the transcriptional programs that drive the anterior-posterior and the dorsal-ventral patterning of the developing brainstem, emphasizing its neuronal diversity. Next, they discuss the transcriptional control that secures the specification of certain groups of neurons that form the central respiratory system in the hindbrain. This discussion includes the pontine groups, and the neurons of both the dorsal and the ventral medullary respiratory columns. Lastly, the authors consider disturbances of TF-coding genes  that cause congenital respiratory syndromes, as a possible path to further understand the genesis, specification, and function of respiratory neurons and the basis of breathing. The review is well-illustrated, and the readers can visit the following references for further details , mid (16–19 GW), and late (23–26 GW) fetal stages. The authors reanalyzed two publicly available single-cell RNA sequencing (scRNA-seq) datasets  is a large brain region that compromises a diversity of neuronal and non-neuronal cell types. The timely appearance and connectivity of these cells during development are crucial for normal PFC anatomy and functions , as these cells were undergoing 8-day neural induction . This epileptic neurodevelopmental disorder is caused by heterozygous variants in the ZEB2 gene, which encodes the TF ZEB2  in exon 8, with those from two healthy donors. The authors used a 65-day protocol that started with neural induction and concluded with GABAergic differentiation  as a framework for further study to better understand the underlying cellular and molecular basis behind the neuropathogenesis and seizures in MWS, that could potentially lead to interventions of the disease.The hiPSC model was also used by Arcuschin et al. contributed to this Research Topic with a mini review that links mechanisms of robustness with neurodevelopment. The robustness of a biological system is defined as its ability to buffer internal and external perturbations, generated by genetic and epigenetic variations, molecular noises or environmental fluctuations, in favor of promoting a reliable output or a particular phenotype , as mechanisms to buffer perturbations during neurodevelopment. In addition, they present miRNAs [e.g., miR-9a for the Senseless TF  is a highly expressed brain water channel that is essential for fluid homeostasis   across the AQP4 gene in both FXTAS and non-FXTAS individuals, by genotyping 160 FMR1 premutation Caucasian carriers . No significant association with the risk of developing FXTAS was found for any of the analyzed SNPs neither for the major and minor allele haplotypes (HTMa and HTMi); conclusion that was maintained after correction by multiple tests. The authors discuss the limitations of their study regarding sample sizes and age differences between FXTAS and non-FXTAS groups. They also stated the need to further study AQPs and the glymphatic system, to either confirm or discard their involvement in FXTAS initiation and its progression.The potential association of FXTAS with variations in the human Bernardinelli et al. present two novel sequence variants of POU3F4: g.5284delA and g.6045T>A, which encode truncated protein products of the TF POU3F4, p.S74Afs*8 and p.C327*, respectively. These mutations were identified by genotyping two male Caucasian subjects, who had been diagnosed with typical hallmarks of POU3F4-related hearing loss [incomplete partition of the cochlea type 3 and enlarged vestibular aqueduct  predicted in the wild type (WT) POU3F4. However, it was noted that the spatial pattern of the nuclear p.C327* was different than that of the WT POU3F4. In addition, both variants failed to properly activate the expression of a reporter gene and to enhance the transcription of POU3F4-driven genes, which were identified by RNA-seq under the influence of the WT TF. Among these genes, the authors selected SLC6A20 for further analysis due to the essential function of SLC6A20 as an amino acid transporter, as well as the association that this class of transporters has with some pathologies, notably hearing loss and cognitive impairment . At the molecular level, the circadian intracellular machinery consists of interlocking transcriptional-translational feedback loops (TTFL) controlled by clock genes (CG), which encode activating and inhibitory TFs [e.g., Clock, Bmal1, Pers and Crys  mouse model  by microRNAs represent a promising avenue in nervous system-related diseases, especially as biomarkers and as prognostic tools (Romano et al., Overall, the original research and review articles of this second volume illustrate the complexity behind a healthy and diseased nervous system, and how TFs and transcription regulation are essential in pacing its development and functions. We expect this Research Topic will encourage researchers to delve deeper into the role of TFs and transcription regulation in homeostatic and adverse conditions. There is potential here to uncover novel biomarkers that could lead to new prognostic tools and new therapeutic remedies.EM: Writing—original draft, Writing—review and editing. VM: Writing—review and editing.


Higher cognition in humans, compared to other primates, is often attributed to an increased brain size, especially forebrain cortical surface area. Brain size is determined through highly orchestrated developmental processes, including neural stem cell proliferation, differentiation, migration, lamination, arborization, and apoptosis. Disruption in these processes often results in either a small  or large  brain. One of the key mechanisms controlling these developmental processes is the spatial and temporal transcriptional regulation of critical genes. In humans, microcephaly is defined as a condition with a significantly smaller head circumference compared to the average head size of a given age and sex group. A growing number of genes are identified as associated with microcephaly, and among them are those involved in transcriptional regulation. In this review, a subset of genes encoding transcription factors , whose functions are important for cortical development and implicated in microcephaly, are discussed. Microcephaly is a clinical condition where the occipito-frontal head circumference (OCF) of an individual is significantly smaller than the average head size of the population for a given sex and age  or secondary  microcephaly. Congenital microcephaly is usually caused by deficiencies in the number of neurons (either reduced production or increased loss), while postnatal microcephaly is more often attributed to defective brain growth and/or neurodegeneration causing brain atrophy  that cause cell cycle-related defects when mutated   genes have been identified  bHLH (basic helix-loop-helix) genes such as MYCN and TCF4; (3) FOX (forkhead box) genes, FOXG1 and FOXR1; (4) SOX (sex-determining regions Y-related HMG box) genes belonging to SOXB1 (SOX2 and SOX3) and SOXC (SOX4 and SOX11) subfamily; and finally (5) zinc finger genes, ZNF238 and ZNF355 , and they are responsible for 55–70% of Coffin–Siris syndrome cases, where microcephaly can manifest as one of the phenotypes  homeobox genes such as d ZNF355 . While nThe homeobox gene family members are characterized by 180-bp DNA sequences known as the “homeobox” encoding a 60-amino acid protein domain called the “homeodomain” . Most hoDrosophila, which resulted in homeotic transformation   implicated in the pathogenesis of microcephaly  belongs to the PRD class, one of the three largest classes of homeobox genes. It is the vertebrate homolog of Drosophila aristaless , essential for the formation of the head segment and the axis specification of appendages , hydranencephaly and abnormal genitalia (HYD-AG), and Proud syndrome   and subventricular zone (SVZ)  and medial ganglionic eminence (MGE) , ne (SVZ) . Howeverne (SVZ) . In contce (MGE)  and remace (MGE) . InteresCdkn1c expression , resulting in premature cell cycle exit and depleting progenitor pools, leading to a reduced brain size. Furthermore, the loss of Arx disrupts PAX6 and TBR2 expression in progenitor cells  class of homeobox gene family. The protein contains a homeodomain (for DNA binding) and two cysteine-rich LIM zinc finger domains (required for zinc binding and functioning as a modular protein-binding interface to mediate protein–protein interactions)  which is essential for wing development , has been shown to act as a selector gene in the developing mouse cerebral cortex, specifying cortical identity in a cell-autonomous fashion while suppressing hippocampal organizer fate   and missense variants in LHX2 from 19 individuals (18 families) presenting with a variable neurodevelopmental phenotype, including microcephaly, intellectual disability, autism spectrum disorder, and other behavioral anomalies. Among 10 patients carrying likely pathogenic variants with occipito-frontal head circumference (OFC) records available, 7 patients showed microcephaly at the time of investigation ; 4 of them had deletions or nonsense variants (likely gene-disrupting), and 3 had missense variants in the LIM domain or homeodomain  for two missense variants located in the homeodomain, impaired interaction with co-factor LDB1 for another variant located in the LIM domain, and impaired transcriptional activation for four missense variants. These data suggest a loss-of-function effect of LHX2. Importantly, the identification of more genetic cases in future will strengthen the argument for the role of LHX2 in microcephaly.Until recently, MEIS2 belongs to the three-amino acid-loop extension (TALE) superfamily of the HOX class of homeobox genes, a homolog of the Drosophila homothorax gene  with a 15q14 deletion presented with microcephaly compared to 2 of 11 patients (18%) with de novo MEIS2 variants  is smaller in null mice when compared to controls  or thyroid-specific enhancer binding protein (T/EBP), belongs to the ANTP class of homeobox genes and is the mammalian homolog of the Drosophila scarecrow (scro)  and thyroid bud, and later in the lung epithelium , congenital hypothyroidism, and infant respiratory distress symptoms  as well as nonsense mutations in NKX2-1 have also been linked to microcephaly that occasionally co-occurs with brain–thyroid–lung syndrome   gene, which regulates anterior patterning as well as brain and eye development  showed genitourinary defects, and two of these six had microcephaly  have reduced cortical size  and the proportion of differentiating neurons was increased  superfamily genes contain two highly conserved and functionally distinct domains: the “basic domain” at the N-terminus and the “HLH domain” at the C-terminus Jones, . The basMYCN is a member of the MYC family of proto-oncogenes and encodes one of the basic helix-loop-helix-zipper (bHLHZ) classes of transcription factors, MYC-N (or N-MYC). The protein forms sequence-specific DNA-binding heterodimers with the bHLHZ protein MAX   are known to cause type I Feingold syndrome (FS1), which is characterized by variable combinations of microcephaly, limb malformation/digit anomalies, intestinal atresia (blockage/obstruction), and mild to moderate intellectual disability  is often confused with the transcription factor 7-like 2 gene , which is downstream of the WNT pathway and referred to as T-cell factor 4 and often mistakenly abbreviated as TCF4. Haploinsufficiency of TCF4 is known to cause Pitt–Hopkins syndrome, which is characterized by developmental delays with severe intellectual disability, dysmorphic facial features, and episodic hyperventilation and/or breath-holding while awake, among other features. Microcephaly has been reported in up to 60% of Pitt–Hopkins syndrome  gene. Head involution is blocked in mutant embryos, causing an alteration of the head exoskeleton    family of transcription factors is named after the ectopic head structures of the FOXG1 was originally named brain factor 1 (BF-1) when it was identified as the Hepatocyte Nuclear Factor 3 (HNF-3) homolog expressed in the developing rat forebrain   compared to MECP2-mediated Rett syndrome, “FOXG1 syndrome” is now considered a distinct disorder . The analysis of newborn mutant mice found cortical thinning with enlarged ventricles  in an individual with severe neurological symptoms, including postnatal microcephaly, progressive brain atrophy, and global developmental delay, has been reported  and a mitochondrial reductase enzyme, DHRS2, each of which plays a role in protective stress response , an essential driver in neurodevelopment, whose dysregulation is associated with microcephaly and other neurodevelopmental syndromes  expression, thus providing cells with protective machinery against environmental stressors. FOXR1 appears to play this role as well, given that its most responsive target genes are two members of the HSP70 family (SOX (sex-determining region Y-related high mobility group box) gene family members encode SOX transcription factors, which belong to the HMG (high mobility group) box superfamily of DNA-binding proteins , with group B further divided into B1 and B2  and were first identified in the screen for homologous genes to the sex-determining gene Sry, which contains an HMG box domain  (HMG-VP16) or EnR (transcription repression domain of the D. melanogaster Engrailed protein) (HMG-EnR) into chick embryos revealed that SOX3 is necessary for the formation of neuroectoderm, maintenance of the neural progenitor state, and suppression of neuronal differentiation  is essential for neuronal differentiation , but some patients also present with microcephaly and other variable phenotypes, including hippocampal abnormalities, epilepsy, and motor problems, as well as dysmorphic facial features and genital anomalies  with characteristic facial features, digits, and eye abnormalities  contain a zinc finger domain that can interact with DNA, RNA, PAR (poly-ADP-ribose), and other proteins and are involved in a wide range of cellular processes such as transcriptional regulation, ubiquitin-mediated protein degradation, signal transduction, actin targeting, DNA repair, and cell migration, among others  and histidine (H) in different combinations   transcription factor containing four zinc finger domains (responsible for DNA binding) and a BTB/POZ domain (multifaceted protein–protein interaction motif) , intermediate zone (IZ), and subventricular zone (SVZ), but not in the marginal zone (MZ). Double-labeling studies with PAX6 and TBR2 showed that the onset of ZNF238 expression coincides with the transition from PAX6-positive cells  to TBR2-positive cells (intermediate progenitor cells), the initial stage of intermediate progenitor cells , ZNF335 (NIF1) encodes a nuclear zinc finger protein known as a coregulator of nuclear hormone signaling and part of the H3K4 methyltransferase complex  and death in all except one  and protein–protein interactions. Genetic variants of these transcription factors cause a spectrum of neurodevelopmental conditions ranging from mild learning disabilities without obvious structural abnormalities of the brain to severe brain malformations  resulting in lethality during early life.In understanding the pathophysiologic mechanisms underlying microcephaly, knockout mouse models have been useful. Accumulating data suggest that transcription factors can control brain growth and size by directly regulating the expression of the target genes that act on cycle progression, cell cycle exit, neurogenesis, or cell survival  or missense mutations in critical domains such as homeodomains tend to give rise to a more severe microcephaly with higher prevalence. Alternatively, it is also possible that these variabilities could arise from the involvement of additional genetic interactors that could affect the severity or prevalence of a transcription factor-associated microcephaly .The knowledge we have built from the studies of individual transcription factors will further our effort to understand brain development in a more systematic way, focusing on the orchestration among all the transcription factors in transcriptional networks. Given that most transcription factors (~75%) work as heterodimers Walhout, , it willYL: Writing – original draft, Writing – review & editing.


These structural studies inform HKMTs’ roles in tumorigenesis and provide the foundations for developing new therapeutic approaches targeting HKMTs in cancers.Histone lysine methyltransferases (HKMTs) deposit methyl groups onto lysine residues on histones and play important roles in regulating chromatin structure and gene expression. The structures and functions of HKMTs have been extensively investigated in recent decades, significantly advancing our understanding of the dynamic regulation of histone methylation. Here, we review the recent progress in structural studies of representative HKMTs in complex with nucleosomes , with emphasis on the molecular mechanisms of nucleosome recognition and  In eukaryotic cells, the nucleosome, which contains four histone proteins  tightly wrapped by 147 base pairs of DNA, is the basic unit of chromatin . DiverseS-adenosylmethionine (AdoMet) to the ε-nitrogen of the lysine residue. There are six well-studied lysine methylation sites in mammalian cells: K4, K9, K27, K36, and K79 on H3 and K20 on H4. Different methylation sites and different methylation levels have distinct biological outcomes, and their combinations significantly increase the complexity of “histone codes” in regulating gene expression . All HKMTs, with one exception (Dot1-family methyltransferases), contain a conserved enzymatic SET  domain initially identified in su(var)3-9, enhancer-of-zeste, and trithorax proteins . The enzreviewed . Here, wWith the rapid development of single-particle cryo-electron microscopy (cryo-EM), more than forty structures of HKMTs in complex with nucleosome core particles (NCPs) have recently been solved , includiSaccharomyces cerevisiae, while mammalian MLL family proteins contain six members, including MLL1 (KMT2A), MLL2 (KMT2B), MLL3 (KMT2C), MLL4 (KMT2D), SETD1A (KMT2F), and SETD1B (KMT2G). MLL-family proteins can methylate H3K4 through their C-terminal catalytic SET domain, but their intrinsic HKMT activities are extremely low  family methyltransferases, which are conserved from yeast to mammals. There is only one member (Set1) in mely low . The HKMmely low . Structumely low , but theMore recently, several cryo-EM structures of MLL1, MLL3, and yeast MLL-homolog COMPASS (complex proteins associated with Set1) in complex with NCP were reported , sheddinNotably, the MLL1 complex could bind unmodified NCP in multiple binding modes (mode 1 and mode 2 in SET and stabilize the arginine-rich motif (ARM) helix of Set1 by fastening it to the nucleosomal acidic patch  is essential for maintaining transcriptional repression and cellular identity . The corChaetomium thermophilum and humans  domain to anchor Set2 on nucleosomes . In contThe NSD family of methyltransferases plays a crucial role in chromatin regulation and is closely linked to oncogenesis . The recDot1 (disruptor of telomeric silencing 1) or DOT1L (Dot1-like) is the only known methyltransferase that can catalyze mono-, di- and tri-methylation of H3K79 in yeast or mammals, respectively . Unlike Dot1 or DOT1L methylates only nucleosome substrates but not histone octamers or H3 . Five grNle , the Wolberger laboratory reported the structure of DOT1L bound to ubNCP in the active state (16KRHR19) in the tail of H4 interacts with the N-terminal region of DOT1L. The H4 tail and ubiquitin clamp the opposing ends of DOT1L to position the DOT1L active site over H3K79  complex is often captured in a poised state, in which the active site of DOT1L is too far from H3K79 for efficient catalysis  and 4D. ve state . Compareve state   is the only known methyltransferase responsible for H4K20 monomethylation and plays pivotal roles in genome replication and stability . The cryomal DNA . SET8 spomal DNA . MutagenNumerous studies have shown that mutations or dysregulations of HKMTs can lead to physiological abnormalities and cause a wide range of human diseases  Bhat et. AccordiMLL1  is a malignant hematological tumor with high incidence and poor prognosis and is caused by rearrangements in the allele of MLL1 . MechaniMLL1 . The remMLL1 . MoreoveMLL1 , 2019. MMLL1 , and MLLleukemia . Unlike ncations . For exancations . MLL3/4 ncations .AS: 3775–3786), which is not conserved among MLL family proteins, extends along the β-propeller surface of WDR5 and interacts with the activation segment of RBBP5 (RBBP5AS: 329–336) through an extensive network of hydrophobic interactions  (16KRHR19) binds to a groove in the N-terminal portion of DOT1L and is also essential for the HKMT activity of DOT1L  , 2013. Ietostat) . Structuetostat) . Furtherof DOT1L  , and malignant peripheral nerve sheath tumors (MPNSTs) . StructuEZH2Y292   and histone methylation  have been elucidated. It is expected that future biochemical and structural studies will uncover more crosstalk between a specific histone methylation and other histone modifications from the same nucleosome and the neighboring nucleosome, highlighting how the “histone code” is synergistically established and interpreted by a combination of histone-modifying enzymes.The H2A–H2B acidic patch creates a highly negatively charged groove on nucleosomes and is a docking “hotspot” for most chromatin-binding proteins, including histone-modifying enzymes, chromatin remodeling complexes, and polymerase complexes . Most HKIt should be noted that the current HKMT-nucleosome structures are all determined at the mononucleosomal or dinucleosomal levels. It will now be essential to extend similar structural studies to nucleosome arrays or chromatin fibers, which are the more physiological substrates for HKMTs. The high-resolution structures of tetranucleosomes and 30 nm chromatin fibers reveal that, in folded chromatin fibers, the nucleosomal H2A–H2B acidic patch is occupied by the H4 tail from the neighboring nucleosome, and this interaction is vital for chromatin compaction . ConsideHKMTs often orchestrate transcription factors, histone chaperones, chromatin remodeling complexes, and non-coding RNAs to regulate nucleosome structure and downstream transcription. How HKMTs collaborate with other factors to establish the epigenetic states of chromatin remains largely unexplored. For example, a recent report found that the MLL-family methyltransferase MLL4 associates with the chromatin remodeling complex BAF , but howGrowing evidence has demonstrated the close association between the misregulation of HKMTs and tumorigenesis , so targ


Genome duplication occurs through the coordinated action of DNA replication and nucleosome assembly at replication forks. Defective nucleosome assembly causes DNA lesions by fork breakage that need to be repaired. In addition, it causes a loss of chromatin integrity. These chromatin alterations can be restored, even though the mechanisms are unknown. Here, we show that the process of chromatin restoration can deal with highly severe chromatin defects induced by the absence of the chaperones CAF1 and Rtt106 or a strong reduction in the pool of available histones, and that this process can be followed by analyzing the topoisomer distribution of the 2µ plasmid. Using this assay, we demonstrate that chromatin restoration is slow and independent of checkpoint activation, whereas it requires the action of transcription and the FACT complex. Therefore, cells are able to “repair” not only DNA lesions but also chromatin alterations associated with defective nucleosome assembly. Nucleosome position is critical for genome regulation, as it dictates DNA accessibility and therefore the processivity of essential processes like transcription, replication, DNA repair and homologous recombination. Accordingly, chromatin alterations may cause from a severe loss of cell fitness to lethality, and are associated with cancer, neurodevelopmental disorders and aging6. More specifically, chromatin assembly defects can lead to replication fork instability, generating DNA lesions that need to be repaired9.The structural unit of chromatin is the nucleosome, formed by 147-bp of DNA wrapped around an octamer of core histones. Nucleosomes are not randomly distributed on the genome but occupying preferential positions with respect to the DNA sequence. These positions are established by the DNA sequence composition and the action of chromatin remodelers, general regulatory factors (GRFs) and transcription10. Replication-coupled (RC) nucleosome assembly involves the action of histone chaperones that interact with replisome components to incorporate both parental and newly synthesized histones into the nascent strands12. In yeast, the chaperone Asf1 presents newly synthesized H3/H4 dimers to the acetyltransferase Rtt109 for its acetylation at H3K56, a modification that enhances the affinity of the dimer for the histone chaperones CAF1  and Rtt10616. These two chaperones play redundant roles in the deposition of new histones, and only the lack of both complexes strongly affects this process16. However, a double mutant lacking CAF1 and Rtt106 is not significantly affected in cell growth, which is explained by the action of the FACT (facilitates chromatin transcription) complex (formed in yeast by the histone chaperones Spt16 and Pob3)18.Nucleosomes are assembled during S phase through a process that is coupled to DNA synthesis, with the first nucleosome deposited ~ 250-bp behind the replication fork19. These chromatin-disruptive activities are required for the advance of the replisome and facilitated by the process of parental histone recycling21. It was early observed that the newly assembled differed from the bulk chromatin24, reflecting the need to recover the original chromatin features in order to maintain genome functionality. This process, termed chromatin maturation, involves both nucleosome repositioning and recovery of parental histone marks diluted by the incorporation of new histones, and has been studied in the last years in yeast, Drosophila and mouse embryonic stem cells (mESCs) using genome-wide approaches that compare nascent and mature chromatin33. These studies have mostly focused on the chromatin organization of the gene units, as both transcription factors (cis and trans) and transcription activity are major determinants of nucleosome positioning34. A conserved feature across eukaryotes is the presence of a nucleosome-free region (NFR) at promoters flanked by two well positioned nucleosomes (− 1 and + 1), and a regularly spaced nucleosome array downstream the + 1 nucleosome with fuzzier positions as nucleosomes are more distant from the promoter35. Chromatin organization at transcription units is lost at nascent chromatin in all analyzed cells, with gaining of nucleosomes at the NFRs and global loss of nucleosome positioning. This suggests that RC-chromatin assembly outcompetes DNA binding proteins, including transcription factors, offering an opportunity to modify chromatin patterns30. The recovery of matured nucleosome patterns at promoters and enhancers in Drosophila and mESCs takes from 30 to 120 min, and at least in mESCs it requires active transcription except for enhancers with nucleosome destabilizing DNA sequences29. In yeast, this process is faster (less than 5 min), does not require transcription (likely due to the presence of AT-rich DNA sequences at its promoters), and is associated with the binding of GRFs32. In contrast to promoters, chromatin maturation at the gene bodies is slower and requires transcription elongation both in yeast and mESCs32.Paradoxically, building up chromatin at nascent strands during DNA replication is associated with the disruption of parental chromatin ahead of the fork36. This likely reflects the capacity of cells to buffer huge oscillations in gene expression and deal with high levels of genetic instability. However, some of the studies on chromatin maturation have shown that cells are able to partially restore chromatin alterations induced by the absence of CAF1, Asf1 or Rtt10937. Here, we have used a plasmid-based topological assay and genome-wide nucleosome profiling to show that cells are able to restore the highly severe loss of chromatin integrity induced in cac1∆ rtt106∆ and histone-depleted mutant cells. Furthermore, we show that chromatin restoration is facilitated by the action of transcription and the FACT complex.Unexpectedly, yeast cells are viable under conditions that cause severe chromatin integrity defects, as the absence of both CAF1 and Rtt016 or a strong reduction in the pool of available histones38. This loss of negative supercoiling is due to the fact that the assembly of each nucleosome introduces one negative superhelical turn39. This topological change can be detected by analyzing the distribution of topoisomers of a plasmid in chloroquine-containing gels, and has been extensively used to address chromatin alterations in vivo and in vitro44. Specifically, the loss of negative supercoiling in histone-depleted yeast cells can be detected by analyzing the endogenous 2µ plasmid in a strain in which the only source of histone H4 is under control of the doxycycline-regulated tet promoter . These repeats can recombine leading to equal amounts of two plasmids that differ in the orientation of one unique region with respect to the other  . It is worth noting that t::HHF2 and cac1∆ rtt106∆ shared similar chromatin alterations; high accessibility of the nucleosomal DNA and similar modified bands.These results suggest that the alterations in chromatin structure induced by defective histone deposition occur transiently during S phase and are post-replicatively restored. To confirm this, we analyzed the pattern of nucleosomes in the 2µ plasmid by indirect-end labelling of MNaseI-treated cells at different times during the cell cycle. We focused on the chromatin structure of an ter Fig. D. These 48, which allows nucleosomes to be mapped along the whole genome. We analyzed the nucleosomal landscape of cac1∆ rtt106∆ and wild type cells both in G1 and G2 phases (60 min after G1 release) to allow completion of genome replication. The absence of CAF1 and Rtt106 during DNA replication caused severe defects in the distribution of nucleosomes in G2  followed by dynamic analysis of nucleosome position and occupancy by sequencing (DANPOS) G2 Fig. A, consis31. In contrast, the cac1∆ rtt106∆ mutant showed a significant drop in the occupancy of nucleosome − 1 in G1 relative to the wild type strain 53. We observed that a triple mutant cac1∆ rtt106∆ mec1∆, defective in DDC activation, was proficient in the recovery of negative supercoiling 54. Since transcription was essential to exit from G1 . After the shift, cells were maintained at restrictive temperature for 90 min. The absence of transcription post-replication did not affect the pattern of plasmid supercoiling during the cell cycle . The loss of negative supercoiling in the triple mutant cac1∆ rtt106∆ rpb1-1 was less pronounced than in the double mutant cac1∆ rtt106∆ , even though a slight recovery was observed in the triple mutant at later times . Therefore, transcription helps to restore the loss of chromatin integrity associated with defective RC-nucleosome assembly.Transcription activity helps to correctly position nucleosomes G1 Fig. A, cells 55. First, we addressed the role of the HIR complex , which has been involved both in chromatin maturation and restauration of cac1∆-associated chromatin defects28. The absence of the HIR complex in cells lacking its major subunit (Hir1) did not prevent the recovery of plasmid negative supercoiling in cac1∆ rtt106∆ cells  and released at semi-permissive temperature (31 °C) until the following G1 phase, which required different times for each strain. The logic behind is to allow a complete restoration of the cac1∆ rtt106∆-induced chromatin defects. At this semi-permissive temperature, plasmid topology was hardly affected in the spt16-G132D mutant  and Drosophila (Caf1-105 knockdown)37, although the mechanisms of restoration are poorly understood. In yeast, asf1∆ and rtt109∆ mutants delay histone deposition due to a lack of H3K56 acetylation that reduces histone delivery to CAF1 and Rtt106. Accordingly, chromatin is less affected in asf1∆ and rtt109∆ than in cac1∆ and rtt106∆ mutants25. Yet, chromatin assembly defects in cac1∆ and rtt106∆ mutants are buffered by the action of Rtt106 and CAF1, respectively16. Here, we have shown by genome-wide MNase-seq that the severe loss of chromatin integrity induced during S phase by the absence of CAF1 and Rtt106 is largely restored post-replication, and that the process of chromatin restoration can be followed by analyzing the level of plasmid supercoiling along the cell cycle. This provides an alternative to the more expensive and time-consuming MNase-seq assay to screen for genetic requirements of the chromatin restoration process. Using this plasmid topology assay, we have shown that cells are able to restore even the severe chromatin defects induced by a strong reduction in the pool of available histones, and that chromatin restoration is facilitated by the action of transcription and the FACT complex. We have focused on the monomeric and not in the multimeric forms of the 2µ plasmid to study the connection between DNA topology and chromatin alterations, thus minimizing template-specific effects. In any case, chromatin dynamics is influenced by the structural and functional particularities of the analyzed regions, and therefore a deeper characterization will require genome-wide approaches.The efficiency of the nucleosome deposition process during DNA replication eliminates chromatin characteristics, which are recovered through a maturation process that depends on DNA sequence composition, GRFs and chromatin remodeling factors, and transcriptioncac1∆ rtt106∆ cells during DNA replication become restored in G1. However, chromatin was more altered in G2 than in S phase, which is consistent with the severe genome-wide chromatin assembly defects remaining in histone-depleted cells arrested in G2/M57. These results suggest that chromatin restoration is slower than chromatin maturation, which is completed in 5–20 min after replication fork passage28, yet highly efficient even under conditions that strongly disrupt the chromatin landscape as those induced in cac1∆ rtt106∆ and t::HHF2 mutants. The accumulation of chromatin alterations and their “repair” during chromatin restoration can also be detected by following the distribution of plasmid topoisomers in RC-nucleosome assembly mutants during the cell cycle. These mutants display a strong and transient loss of plasmid negative supercoiling during the cell cycle as a consequence of RC-chromatin disruption. In contrast, the population of plasmid topoisomers does not change in the wild type, which reflects the speed and efficiency of the chromatin maturation process. Therefore, this assay allows to specifically follow chromatin restoration.Our genome-wide analysis shows that most chromatin assembly defects generated in 53. We show that this arrest is not required for chromatin restauration, although it likely provides time to coordinate this process with the repair of the DNA lesions that will activate the checkpoints.A common feature of chromatin assembly mutants is a metaphase arrest triggered by the activation of the DDC and/or SAC34, and it is required for chromatin maturation after DNA replication32. We show that transcription facilitates chromatin restauration. The genome-wide chromatin analysis showed that the loss of nucleosome phasing at G2 in the cac1∆ rtt106∆ mutant mainly affected the gene bodies, as previously observed in histone-depleted cells in G2/M57. The analysis of nascent chromatin at early time points in a cac1∆ mutant showed nucleosome defects both at promoters  and gene bodies (loss of phasing)28. Therefore, the promoter architecture of the cac1∆ rtt106∆ mutant is likely first reconstructed to prime active transcription and restore chromatin in the gene body as proposed for chromatin maturation in yeast, where the rapid binding of GRFs at promoters generate molecular landmarks that fix the positions of flanking nucleosomes30. This mechanism is also likely necessary during the restoration of a severely altered chromatin in order to provide a rule for the transcription machinery to properly reposition nucleosomes during elongation. However, it is unlikely that this process resembles chromatin maturation in the initial steps. During chromatin maturation, restructured promoters with bound GRFs are still refractory to RNAPII recruitment31, which explains why transcription is buffered for a while after replication59. In contrast, defective chromatin assembly in asf1∆ and cac1∆ rtt106∆ cells causes a transient accumulation of aberrant coding and non-coding transcripts behind the replication forks37. This effect is more pronounced in the cac1∆ rtt106∆ mutant, likely because of its higher nucleosome deposition defects and the role of Rtt106 in preventing aberrant transcription50. We speculate that transcription from spurious initiation sites may slow the process of chromatin restoration because of the repositioning of nucleosomes without a correctly defined reference.Transcription activity is a major determinant of nucleosome position27. Less clear is the relationship with transcription for the HIR complex27, a chromatin remodeler that participates in replication-independent histone turnover, preferentially at intergenic regions61. The study of bulk nucleosome organization has also pointed to a role for the HIR complex in the restoration of cac1∆-induced nucleosome assembly defects28. Our plasmid topology assay did not reveal any role for the HIR complex in chromatin restoration in the cac1∆ rtt106∆ mutant. Although the difference might be plasmid-specific, it cannot be excluded that the loss of nucleosome phasing in cac1∆ hir1∆ cells reflects an additive effect of the absence of both complexes, as the hir1∆ mutant by itself displayed a reduction in the amplitude of the nucleosomal oscillation on coding regions28.The requirement of transcription elongation for chromatin maturation supports a role for chromatin remodeling complexes traveling with RNAPII like CHD1 and ISW1b. In agreement with this possibility, nascent chromatin-associated alterations persist in the absence of these chromatin remodelers66. We have observed that the spt16-G132D mutant has no defects in the distribution of plasmid topoisomers but prevents the recovery of the negative supercoiling level lost during DNA replication in a cac1∆ rtt106∆ mutant at semi-permissive temperature (31 °C). Therefore, the amount of Spt16 at this temperature seems to be sufficient to avoid a loss of nucleosomes during transcription but not to restore defective chromatin assembly. This suggests that the mechanism by which FACT restores chromatin is either different or requires more Spt16 than the mechanism by which FACT redeposits nucleosomes during transcriptional elongation. FACT is targeted to chromatin by recognizing the surface of disrupted nucleosomes generated mainly—but not exclusively –by transcription68. This observation, together with the ability of FACT to assemble nucleosomes led to Formosa and Winston to propose a role for FACT in the “repair” of disrupted nucleosomes69. It is likely that the dependency on transcription of the chromatin restoration process in cac1∆ rtt106∆ cells reflects the need to disrupt nucleosomes to target FACT, which would be required at higher levels than in the wild type strain to additionally cope with displaced nucleosomes. An alternative but not exclusive possibility for the higher demand of Spt16 during chromatin restoration is that not only the position but also the integrity of some nucleosomes become affected in cac1∆ rtt106∆ cells, targeting FACT in a transcription-independent manner.FACT is a nucleosome remodeler complex with a critical role in nucleosome repositioning during transcription elongation. FACT travels with the RNAPII promoting the redeposition behind RNAPII of the original nucleosomes evicted during elongation through a stepwise mechanism of nucleosome disassembly-assembly that helps to maintain the epigenetic identityIn summary, cells are able to largely restore a severe loss of chromatin integrity induced under conditions of defective nucleosome assembly, providing a mechanism to buffer its impact on cell fitness. In addition, using plasmid topology as an easy and specific assay to study chromatin restoration, we have shown that this process requires the action of both transcription and FACT. This assay may help to uncover additional factors involved in chromatin restoration, as a previous step to a more detailed genome-wide characterization.rpb1-1, spt16 and Gp::CDC6 strains) or supplemented minimal medium (SMM) (rest). For metaphase synchronization, cells were treated with 15 µg/ml nocodazole for 1 h. For G1 synchronization, cells were grown to mid-log-phase and α factor was added twice at 90 min intervals at 0.5 μg/ml, except for t::HHF2 strains (treated with 1 μg/ml) and rpb1-1 strains . Cells were then washed three times and released into fresh medium with 50 μg/ml pronase. For G1 resynchronization, cells released into S phase were washed and resuspended in fresh medium with α-factor at 1 μg/ml (t::HHF2 strains) or 0.5 μg/ml (rest of strains) until G1. To induce nucleosome depletion, t::HHF2 cells growing in the presence of 5 µg/ml doxycycline were shifted to 0.25 μg/ml during G1 synchronization and release. Cdc6 depletion was performed as previously described70. Briefly, Gp::CDC6 cells were synchronized in metaphase in 2% galactose-containing medium with 1% DMSO and 15 µg/ml nocodazole for 2 h, shifted to 2% glucose-containing medium with DMSO and nocodazole for 2 additional hours, synchronized in G1 in 2% glucose-containing medium with α-factor for 2 h, and released into fresh 2% glucose-containing medium with 50 µg/ml pronase for 1 h.Yeast strains used in this study are listed in Table 36. Cells were fixed with 70% ethanol, washed with phosphate-buffered saline (PBS 1X), incubated with 1 mg of RNaseA/ml PBS, and stained with 5 µg/ml propidium iodide. Samples were sonicated to separate single cells and analysed in a FACSCalibur flow cytometer.DNA content analysis was performed by flow cytometry as reported previously36. Briefly, total DNA was extracted using a zymolyase-SDS standard protocol and run into 0.8% TPE 1 × agarose gels containing 4 μg/ml chloroquine for 36 h at 1.6 V/cm with recircularization. Negatively supercoiled topoisomers are resolved at this chloroquine concentration. Gels were blotted onto Hybond™-XL membranes and hybridized with a 32P-labeled FLP1 fragment amplified by PCR from genomic DNA with oligos 5′-tgattacacataacggaaca-3′ and 5′-ttcagcactaccctttagc-3′. Signals were acquired in a Fuji FLA5100 and quantified with the ImageGauge analysis program. The total DNA signal (area under the curve) of the raw topoisomer profiles was equalized to eliminate DNA loading differences.The distribution of topoisomers of the 2µ plasmid was performed as previously described57. Briefly, cells were fixed for 15 min with 1% formaldehyde. Glycine was added to quench the reaction at a final concentration of 125 mM. Cells were sedimented, washed twice with cold PBS and stored at − 80 °C until use. Extracts for MNase digestion were resuspended in 1 M sorbitol/50 mM Tris HCl and digested for 1 h at 30 °C with 4.5 mg of zymoliase 20 T (AmsBio 120491-1) in gentle shaking. Samples were washed first with cold solution I  and then with cold solution I plus 0.1 mM PMSF, then resuspended gently in cold solution II , and finally treated for 20 min at 37 °C with different concentrations of MNase (SIGMA N3755). The reaction was then stopped by adding 3 mM EDTA/4 mM Tris HCl and 10% SDS. To revert crosslinking, samples were incubated for 90 min at 37 °C with 1.5 mg of proteinase K and then overnight at 65 °C. DNA was extracted from samples using a standard phenol–chloroform extraction, treated with RNase A and loaded in a 1% agarose gel to check MNase digestion. MNase digestions used for indirect end labelling were incubated with EcoRI, resolved in 1.5% agarose gels, blotted onto a HybondTM-XL membrane and probed with a 32P-labeled specific PCR fragment located close to one of the EcoRI sites (oligos 5′- ataccaattcctcttcctag-3′ and 5′-tccaaatatacaagtggatc-3′). Signals were acquired in a Fuji FLA5100 with the Image Gauge analysis program.Chromatin analyses by MNaseI and indirect-end labeling were performed as previously described57. Briefly, MNaseI–digested DNA samples from two (G1 and G2) or one (S phase) biological replicates for each yeast strain were obtained as previously indicated for indirect-end labelling. MNase digested samples enriched in mononucleosomes were loaded in a 1% agarose gel, and the DNA corresponding to mononucleosomes was purified with a DNA purification kit . The DNA size and quality was confirmed by an electropherogram analysis . Library construction and sequencing was performed at Genomics Core Facility of CABIMER. DNA libraries were prepared from 10 ng mononucleosome DNA using the TruSeq Chip Library Preparation kit (Illumina), and the size distribution and molarity of each library were analyzed with the Agilent™ DNA High Sensitivity Kit . DNA libraries were sequenced on the NextSeq 500 Sequencing System (Illumina), and raw data were processed for basecalling, filtering and trimming to generate the FASTQ files using the BaseSpace Onsite v3.22.91.158 Software de Illumina. Sequence reads were mapped to S. cerevisiae genome sacCer3 by BowTie271, and potential PCR duplicates were removed by SAM Tools on the Galaxy platform 72. The peak-calling algorithm Dpos function (DANPOS 2.2.0)73 was used for nucleosome occupancy maps and comparative analyses using default parameters. Average nucleosome occupancy patterns flanking transcription start sites (TSS) from one 73.Chromatin analyses by MNase-seq were performed as previously describedone Fig. A or two  G1 and G or one 75 and resolved on a 8% SDS-PAGE. Rad53 was detected by standard western blot analysis with the rabbit polyclonal antibody JDI4876.Yeast protein extracts were prepared using the TCA protocolSupplementary Information.


Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here we determine the roles of CHD4 to enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility at transcription factor binding sites; its depletion leads to altered motif scanning and redistribution of transcription factors to sites not previously occupied. During GATA3-mediated cellular reprogramming, CHD4 activity is necessary to prevent inappropriate chromatin opening and enhancer licensing. Mechanistically, CHD4 competes with transcription factor-DNA interaction by promoting nucleosome positioning over binding motifs. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents inappropriate gene expression by editing binding site selection by transcription factors. Establishment of an active enhancer is hypothesized to be a multi-step process involving alterations to local chromatin by chromatin remodeling enzymes, histone replacement, and editing of histone and DNA modification 4. All downstream processes flow from the initial event – recognition of DNA sequence by a sequence-specific DNA binding transcription factor. In most cases, TFs are present on the order of thousands to tens of thousands of molecules per cell. In contrast, many transcription factor motifs are present on the order of millions of copies per genome. How TFs are directed to activate enhancers in all the correct locations and only the correct locations is critical, as both failure to activate appropriate sets of genes as well as inappropriate gene activation is typically deleterious to the biological program. Cooperative binding by multiple TFs, partial motif recognition, nucleosome positioning, and chromatin remodeling factors are thought to be involved in selective enhancer formation 7. However, the molecular mechanisms remain elusive.During the cell fate transitions integral to development or transcription factor-dependent cellular reprogramming, lineage-determining transcription factors (TFs) must contend with chromatin to nucleate active enhancers. Some transcription factors such as pioneer factors utilize their intrinsic ability to bind nucleosomal DNA in closed chromatin as a first step in the induction of chromatin opening 9. CHD4 is involved in multiple developmental processes including neural development and cardiac development 12. Similar to other chromatin remodeling factors, genetic and epigenetic data in cancer patients detected frequent alterations in the CHD4 gene suggesting key roles of CHD4 during tumorigenesis and tumor progression 16. Because the NuRD complex contains histone deacetylases such as HDAC1 or HDAC2, it has been thought to act as a gene silencer 20. However, the genomic distribution of CHD4 is predominantly enriched at promoters or open chromatin regions, and the CHD4 function appears to be cell context specific 24.CHD4 (Chromodomain Helicase DNA Binding Protein 4) is a catalytic core component of the NuRD (Nucleosome Remodeling and Deacetylase) chromatin remodeling complex and is known to regulate gene expression and DNA damage responses 25, CHD4 maintains chromatin architecture mainly at intergenic regions. In the absence of CHD4, chromatin sensitivity was increased at closed chromatin, especially at GATA3 binding peaks. Abnormal chromatin opening led to increased expression of genes unrelated to MET. High-resolution nucleosome mapping suggested that CHD4 prevents unnecessary gene expression by mediating nucleosome formation over the GATA3 binding sites. These results demonstrate that CHD4, and by extension NuRD complex, monitors transcription factor/chromatin interactions and modulates gene regulation by proofreading transcription factor site selection for enhancer nucleation.In this study, we measured the impacts of CHD4 depletion in MDA-MB-231 basal breast cancer cells. Characterization of chromatin features by multi-omics methodologies revealed differential impacts of CHD4 knockdown in steady-state cells vs cellular reprogramming processes. CHD4 knockdown in steady-state MDA-MB-231 cells tends to increase chromatin accessibility at promoter-distal regions. Mechanistically, AP1 family transcription factors are involved in altered chromatin binding accessibility and transcription. During the GATA3-induced MET cell reprogramming processes 27. The physical location of most ATAC-seq peaks in the genome was unchanged, while approximately 1/3 of detected peaks were either lost or gained following CHD4 depletion  were used to identify significantly altered transcripts. We identified a large percentage of the transcriptome with significant alterations in steady state transcript level – 1880 genes were upregulated and 1072 genes were downregulated . The numWe observed alterations in the dynamic competition between nucleosomes and transcription factors for DNA upon depletion of CHD4. However, we also observed changes in expression of transcription factor family members, potentially complicating analysis. Therefore, we moved to a more defined system with a temporal component, transcription-factor dependent cellular reprogramming. We established a doxycycline-inducible GATA3 expression system in MDA-MB-231 mesenchymal breast cancer cells. We selected a cell clone that shows minimal GATA3 expression in the absence of doxycycline (hereafter DOX) but expresses biologically relevant amounts of GATA3 protein upon DOX treatment. In this cell clone, GATA3 protein expression was observed within 3 hours after addition of DOX to media and was saturated by 12 hours . Stable 25, transcripts with altered levels were enriched in categories involved in mesenchymal to epithelial transition , loci where GATA3 binding occurs within transposase accessible chromatin (constitutively accessible) and loci where GATA3 binds to inaccessible chromatin that remains inaccessible (constitutively inaccessible) following GATA3 expression  – 3c. RNWhen CHD4 was depleted in this system, we observed striking alterations in several features. Of loci exhibiting significant alterations in ATAC sensitivity, 82% (4548 of 5558) demonstrated an increase in accessibility , similar30. Placenta related genes such as VSTM5, NDNF, HAPLN1 were up-regulated in the CHD4 knockdown cells , nucleosome depletion was observed at the center of GATA3 binding peaks, while nucleosome repositioning and accumulation were observed at the constitutively inaccessible GATA3 binding peaks. To understand the impact of CHD4 depletion on nucleosome remodeling following induction of GATA3, we performed MNase-seq to map nucleosomes during the GATA3-mediated cellular reprogramming. Newly accessible peaks exhibit the characteristic pattern of MNase-resistant, positioned nucleosomes flanking a moderately resistant region centered over the transcription factor binding motif , more than 7 million loci contain the GATA3 consensus motif. In most cases, TFs like GATA3 are present on the order of thousands to tens of thousands of molecules per nucleus, meaning there are roughly 100-fold more potential binding sites than TFs.TFs face multiple challenges in establishing new gene regulatory networks during development, in response to physiologic or environmental signals, and during in vitro cellular reprogramming. These proteins must find appropriate recognition motifs within the genome and they also must contend with physical barriers including chromatin. In eukaryotes with large genomes, including humans, TFs with short recognition motifs must find the correct loci, and only the correct loci, amongst the potentially millions of matches to their binding motif. In theory, degenerate hexameric binding motifs, such as the WGATAR consensus binding motif for GATA3 33. Within the context of a nucleosome, the rotational and translational phasing of DNA necessarily obscures some chemical information where it closely approximates the histone octamer surface, making it unavailable for sequence readers 5. Biochemical and structural data indicate that location of binding motifs near the center versus near the periphery of a nucleosome has a strong influence on binding and stability 35. Higher order structural features of chromatin, including linker histones, assembly into heterochromatin, or partitioning into low contact frequency nuclear compartments are likely to further restrict the available sequence space to be searched by TFs 37. Clearly, other factors must contribute to narrowing the search space and increasing the probability that cellular signals will result in activation (or repression) of the correct subset of genes 38.Chromatin represents a first-line barrier to inappropriate binding of TFs, and it presents a barrier in multiple ways. A subset of TFs cannot read DNA sequence and bind productively at all to DNA wrapped around a histone octamer 40. However, genome-wide mapping finds frequent localization of CHD4 at active gene promoters and enhancers 42, clouding understanding of mechanistic roles by which CHD4 regulates gene expression. In this study, we investigated the roles of CHD4 in steady state basal breast cancer cells and during mesenchymal-to-epithelial transition (MET). In the steady state, CHD4 depletion resulted in a primarily increased chromatin accessibility leading to abnormal gene expression unrelated to breast cancer cell identity. While the loci with decreased chromatin accessibility by CHD4 depletion are enriched at promoters, increased chromatin accessibility regions are enriched at intergenic regions. Motif analysis at differential peaks suggested that CHD4 modulates chromatin binding of multiple AP1 family members. In fact, when CHD4 was depleted, at least three AP1 family proteins, JUNB, ATF3, and FRA1, were redistributed, and enrichment at consensus motif-containing regions was reduced.CHD4 is important for maintenance of chromatin architecture and is known to suppress gene expression during tissue development, cell differentiation, and cell reprogramming 42 where it is integral to the process of enhancer decommissioning during development 41 and reprogramming 40. We propose that NuRD acts, in part, to antagonize transcription factor driven increases in chromatin accessibility – regardless of the ultimate outcome. It seems plausible that local translational motion of nucleosomes relative to transcription factor binding motifs leads to architectural obstacles to motif recognition. At loci that become accessible, co-binding of multiple TFs or recruitment of other chromatin modification/remodeling enzymes generates a competition between factors promoting and opposing DNA accessibility that ultimately reaches a dynamic equilibrium in which some alleles within the population are accessible to structural probes. At loci that fail to become accessible, failure to recruit activating co-factors leads to generation of a very different type of equilibrium, one in which nucleosome translational position relative to the GATA motif permits GATA3 binding to nucleosomal DNA in the absence of detectable accessibility. In this manner, choice of binding sites within the genome can be ‘proof-read’ by chromatin remodelers simply through enforcing a highly dynamic state 44. In principle, such chromatin dynamics would promote two critical outcomes: they would provide assurance against inappropriate transcriptional activation following transcription factor binding at incorrect sites and they would provide an opportunity for rapid decommissioning of enhancers to enable progression to new patterns of gene expression consistent with cellular needs.During GATA3-mediated MET cell reprogramming, CHD4 knockdown again resulted in largely chromatin opening. At both newly accessible and constitutively inaccessible GATA3 bound loci we observed evidence for alterations in local nucleosome positioning, consistent with active roles for chromatin remodelers. Further investigation revealed that depletion of the chromatin remodeling enzyme CHD4, a core subunit of the NuRD complex, dramatically altered this outcome leading to new accessibility at previously inaccessible loci and increasing accessibility to transposition at accessible loci. This abnormal chromatin opening, not observed in the presence of CHD4, was associated with altered gene expression and affected cell fate transition at the cellular level. We speculate that our observations reflect a general property attributable to CHD4, and by extension to NuRD complex. NuRD is found with high frequency at enhancers MDA-MB-231 cells were originally obtained from ATCC. MDA-MB-231 cells were cultured in DMEM high-glucose medium with 10% FBS (Thermo Fisher Scientific or R&D Systems). Doxycycline-inducible GATA3 expression system in MDA-MB-231 cells was developed by lentiviral transduction. Ty1-tagged GATA3 gene was inserted into the pINDUCER20 vector, and the lentivirus was generated by the 2nd generation lentiviral plasmids system using 293T cells. pINDUCER20 was a gift from Guang Hu (NIEHS/NIH). psPAX2 and pMD2.G were gifts from Didier Trono . After antibiotic selection with G418, cell colonies were collected, and GATA3 expression levels were investigated by western blot. The cell clone that has low basal GATA3 expression (without DOX treatment) was used in this study.47. Briefly, DOX treated cells were fixed at 12 hours with 1% formaldehyde. Fixed cells were treated with hypotonic buffer containing 10 mM HEPES-NaOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10% glycerol, 0.5% Triton X-100, and protease inhibitor cocktail (Thermo Fisher Scientific). Chromatin was digested by sonication with Covaris S220 in the lysis buffer containing 20 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 5mM sodium butyrate, 0.1% SDS, and protease inhibitor cocktail. 2.5 μg of each antibody was added to each chromatin solution (1 million cells/reaction). After overnight incubation, protein A/G mixed Dynabeads were added, and the samples were rotated for 2 hours. Eluted DNAs were reverse crosslinked at 65 °C for 4 hours, followed by the incubation with proteinase K for 1 hour and purified by AMPure XP (Beckman Coulter).ChIP-seq libraries were prepared as previously described ChIP-seq libraries were generated by the NEXTflex Rapid DNA-seq kit (PerkinElmer) and sequenced on NextSeq 500  at the NIEHS Epigenomics Core Facility. The same data processing protocol to ATAC-seq was used. Mapped reads were converted to a single fragment and used to generate genome coverage tracks on the UCSC Genome Browser and for metaplot analyses.48 with edgeR.To carry out correlation and differential analysis, we used read counts from the reference peak sets and processed the data using the SARTools pipeline The pGIPZ vectors and lentiviruses encoding CHD4 shRNA and control shRNA were provided by the NIEHS Epigenomics Core and Viral Vector Core. 400,000 cells were plated on 6 cm dishes, and infected with these shRNA lentiviruses at 24 hours and 32 hours after plating. After overnight incubation, the medium was replaced with 4 ml of fresh medium and further incubated for 2 days. The GATA3 expression was initiated by adding DOX  in fresh medium. 12 hours after induction, cells were harvested and resuspended in PBS(−).47. 25,000 cells (in 25 ul) were transferred to new tubes, and nuclei were isolated with CSK buffer . Nuclei were treated with 2.5 μl of Tn5 Transposase (Illumina) in the standard tagmentation reaction buffer (25 μl). The total of 8 PCR cycles were performed to amplify the DNA fragments, and the libraries were sequenced on NextSeq 500 at the NIEHS Epigenomics Core Facility. The raw sequence reads were filtered based on the mean base quality score >20. Adapter sequences were removed by Trim Galorre! (Babraham Institute). Processed reads were mapped to hg19 genome using Bowtie 0.12.8 49, and uniquely mapped reads (non-duplicate reads) were used for the subsequent analysis.The ATAC-seq libraries were prepared as previously described Peak classification was conducted based on the following criteria.Newly accessible peaks are defined as the GATA3 peaks that show (1) >2-fold increase in ATAC-seq signals at GATA3 peaks (+/− 200 bp from peak center) compared to the control (Time 0h) condition and (2) >30 normalized reads at the peak flanking (+/− 1 kbp) region.Constitutively accessible peaks are defined as the GATA3 peaks that show >30 normalized reads at the flanking regions (+/− 1 kbp).Constitutively inaccessible peaks are defined as the GATA3 peaks that show <30 normalized reads at the flanking regions.31. Biotinylated RNA probes were designed and purchased via SureSelect Custom DNA Target Enrichment Probes system with the following probe design specification . The target regions are listed on Supplementary table 1.Capture MNase-seq was performed as previously described Nucleosomal fragments were prepared by digesting nuclei with Micrococcal nuclease (MNase). Sequencing libraries were prepared by NEXTflex Rapid DNA-Seq kit (PerkinElmer). Libraries from different conditions were pooled, and 750 ng DNAs were used to perform nucleosomal DNA fragment enrichment at a subset of GATA3 peaks with the SureSelect Enrichment kit. After the fragment enrichment by RNA probe hybridization and streptavidin pull-down, captured DNAs were amplified by PCR (12 cycles) and sequenced on NextSeq 500 at the NIEHS Epigenomics Core Facility. The same data processing protocol to ATA-seq was used for the capture MNase-seq data but duplicate reads were retained. To generate heatmaps, midpoints only from mono-nucleosomal fragments (120-170 bp) were collected. The midpoint frequency (considered as dyad frequency) was normalized by the data from Time 0.50. Subread featureCounts (version 1.5.0-0-p1) and DESeq2 (v1.10.1) with FDR < 0.01 were used to define differentially expressed genes (DEGs) 51. Pathway analysis was performed by DAVID database 46 using DEGs that have |log2 (fold change)| >0.5. DEGs are summarized in Supplementary Table 2.Total RNAs were purified by the Qiagen RNeasy kit. Sequencing libraries were generated by TruSeq RNA library preparation kit with Ribo-Zero and sequenced on NextSeq 500 (Illumina) at the NIEHS Epigenomics Core Facility. Filtered read pairs were mapped to hg19 by STAR (version 2.5) with the following parameters: -- out-SAMattrIHstart 0 --outFilterType BySJout --alignSJoverhangMin 8 -- outMultimapperOrder Random The control shRNA or CHD4 shRNA expressing MDA-MB-231 cells were prepared as described above using the lentiviruses. The GATA3-induced cells were culture for at least 2 days. 4 million were seeded in each 6-well plate and grown overnight. The confluent cells were scratched with a 1 ml micropipette tip. After scratching, the cells were washed with 2 ml of pre-warmed serum-free medium, and the same volume of the serum-free medium was added to each well. Cell images were taken at multiple time points  by Olympus IX71 microscope, and imageJ (Version: 2.1.0/1.53c) was used to quantify wound areas.52.Anti-Ty1 antibody , JUNB , ATF3 , FRA1 , and CHD4 Abcam  was used for ChIP-seq and western blot. GATA3 antibody was generated in rabbits using recombinant 6x histidine tag-fused GATA3 full-length wild-type protein 25.Data generated in this study are available at Gene Expression Omnibus under Accession Number GSE201797, GSE201798, GSE201799, GSE201800, and GSE201801. GATA3 ChIP-seq data in the stable cell line (GSE72141) were previously generated 1


A considerable part of many eukaryotic genomes consists of repetitive sequences derived from transposable elements (TEs). TEs are genetic elements that have, or once had, the capability to hop around their host genomes. Through their mobility, they threaten host genomic stability; consistently, uncontrolled TE transposition has been linked to infertility and to diseases such as cancer, hemophilia, and neurodegeneration . An incrAt the same time, however, TEs can positively impact their host genome. They act as drivers of genomic evolution, influence neighbouring gene expression and have been suggested to shape developmental processes. For example, during Zygotic Genome Activation (ZGA) in early mouse embryonic development, the expression of hundreds of mouse endogenous retroviruses (MERV) is induced. It has been shown that MERVs provide alternative promoter sequences that activate zygotic genes . More reAnsaloni and colleagues, used multiomic approaches to identify a set of evolutionary young LINE1 whose transcripts are present in the oocyte and which are poised for transcription during ZGA. Through a bioinformatic approach, they attribute their transcription to YY1 activity, paving the way to new research aimed at experimentally validating the role of YY1 in their regulation. Importantly, this work reveals a specific, spatial compartmentalization of MERVs transcription from gene-dense genomic regions and LINE1 from intergenic regions that could coordinately influence the progress of ZGA.In a study published in this special Research Topic, Ma et al., focuses on the various ways in which transposable element (TE) expression and activity affect hPSCs, epigenetic-regulated mechanisms that prevent TE expression in hPSCs, and links between the dysregulation of these mechanisms and human diseases including cancer and neurodegenerative disease. While TEs are largely repressed in most cell types, TE expression and activity is high in hPSCs, and the authors present interesting hypotheses on why this phenomenon occurs. Furthermore, TE sequences influence transcription of both coding and non-coding RNAs in mammalian PSCs, through several different mechanisms, thus impacting PSC homeostasis, pluripotency, and self-renewal. Finally, the authors discuss arguments for the potential contribution of TE sequence fragments in PSCs to tumor development and summarize findings on the epigenetic deregulation of LINE-1 and the endogenous retrovirus, HERV-K in tumor cells and neural cell types present in patients with neurodegenerative disorders.Human pluripotent stem cells (hPSCs) are undifferentiated, self-renewing cells that are present very early after conception, and which can give rise to all differentiated adult cell types. In this special Research Topic, the review titled “Transposable Elements in Pluripotent Stem Cells and Human Disease,” by Drosophila (Another developmental time in which TEs are de-silenced is during germ cell specification when animals epigenetically reprogram their genome to ‘reset’ development. While this is an opportunity for TEs to propagate, it is also a threat to the host progeny’s genome integrity. Taming TE transcription is, therefore, essential for fertility. Eukaryotic genomes have evolved specific molecular machineries to silence TEs transcriptionally and post-transcriptionally. They rely, among others, on the small RNA silencing machinery, DNA transcription factors and chromatin-associated enzymes. One of the chromatin factors involved in TE silencing is the histone demethylase LSD1/KDM1A. Depletion of LSD1 activity induces an increase in the transcription of many TE families in mice  humans , and Droosophila .Ren and colleagues, examine the role of Caenorhabditis elegans AMX-1, one of the three worm homologs of the human LSD1 family, in fertility. They report that worms lacking AMX-1 can produce a small number of nonviable eggs. The sterility observed upon AMX-1 depletion is dependent on p53/Cep1 and correlates with an increase in expression of Tc1 and Tc3 transposons but is not dependent on H3K4me2 accumulation. The authors argue that AMX-1 and another LSD1 ortholog, SPR-5, are functionally non-redundant in the germline, and the sterility observed in AMX-1-depleted animals is linked to p53-dependent TE activation. These findings necessitate future studies aimed at validating this hypothesis through genome-wide and genetic approaches.In this Research Topic, Emmanuelle Lerat summarizes these challenges and provides an overview of the current approaches. She thereby provides a concise list of TE-centric, bioinformatic tools along with brief explanations of their unique features, advantages, and disadvantages.The repetitive nature and frequent mutations within TE families remain a significant challenge in interpreting genome-wide approaches. Despite a continuous effort in developing new tools that would allow us to reliably map and interpret reads from next-generation sequencing-based approaches, we are still lacking a universal and broadly applicable tool set to interpret transcript levels, chromatin modifications, or protein binding at individual copies of repetitive elements. In this special Research Topic, Many outstanding questions remain to be answered in the field: What are the mechanisms by which TEs are regulated throughout the lifespan of individuals? Which TEs are expressed and when? Do TEs have specific roles in development and in diseases? As research in the field of transposable elements continues to advance, addressing these outstanding questions will provide valuable insights into the roles, regulation, and impacts of TEs on genome evolution, development, and disease.


Transcription commonly occurs in bursts, with alternating productive (ON) and quiescent (OFF) periods, governing mRNA production rates. Yet, how transcription is regulated through bursting dynamics remains unresolved. In this study, we conduct real-time measurements of endogenous transcriptional bursting with single-mRNA sensitivity. Leveraging the diverse transcriptional activities in early fly embryos, we uncover stringent relationships between bursting parameters. Specifically, we find that the durations of ON and OFF periods are linked. Regardless of the developmental stage or body-axis position, gene activity levels predict the average ON and OFF periods of individual alleles. Lowly transcribing alleles predominantly modulate OFF durations (burst frequency), while highly transcribing alleles primarily tune ON durations (burst size). Importantly, these relationships persist even under perturbation of cis-regulatory elements or trans-factors. This suggests a novel mechanistic constraint governing bursting dynamics rather than a modular control of distinct parameters by distinct regulatory processes. Our study provides a foundation for future investigations into the molecular mechanisms underpinning spatiotemporal transcriptional control. Eukaryotic transcriptional regulation is an inherently dynamic and stochastic process, orchestrated by a series of molecular events governing productive transcription initiation by individual RNA polymerases (Pol II complexes) , 2. ThisResearch across diverse biological systems, from yeast to mammalian cells, has revealed that transcription occurs in bursts. These bursts entail the release of multiple Pol II complexes during an active phase, often referred to as the “ON” period, followed by a quiescent “OFF” period –9. Howevcis-regulatory elements, nucleosome occupancy, histone modification, Pol II pausing, and enhancer–promoter interactions, may influence distinct aspects of bursting dynamics [One hypothesis that has emerged from previous work suggests that different regulatory factors, including transcription factor (TF) binding, dynamics –19. For dynamics –22. HoweDrosophila embryos. This finding motivated our efforts to perform highly-sensitive live transcription measurements, enabling us to directly measure bursting dynamics without relying on model-specific kinetic assumptions.In our previous study , relyingcis-and trans-regulatory factors. Surprisingly, all data collapse onto strict relationships between bursting parameters. We find that transcriptional activity is primarily determined by the probability of an allele being in the ON period, while the initiation rate remains largely constant. Moreover, we identify a tight relationship between the durations of ON and OFF periods, implying that these periods are not tuned independently. This discovery hints at the existence of a novel mechanistic constraint on the transcription initiation process.Here, we present real-time endogenous transcription measurements with single mRNA sensitivity in developing fly embryos, where tightly regulated spatiotemporal transcriptional dynamics play a pivotal role in determining cell fate. Transcription of the examined genes is monitored across developmental time in addition to body-axis positions, as well as and under perturbation to A corollary of the observed relationship is that lowly transcribing alleles increase activity by predominantly shortening OFF periods, increasing burst frequency, while medium-to-high transcribing alleles predominantly extend ON periods, thereby increasing burst size. Importantly, perturbations to transcription prompt a re-examination of our mechanistic understanding, as these highlight that activity levels, rather than specific regulatory determinants (such as enhancer sequences or transcription factor concentrations), can predict bursting parameters. Overall, our study reveals constraints on the molecular implementation of transcriptional dynamics, thereby guiding future mechanistic investigations.Drosophila embryos. To achieve this we utilized a versatile CRISPR-based scheme [We developed a quantitative approach to measure endogenous bursting dynamics at a single allele level in living d scheme  to incord scheme –29. We er(t)  . ImportaKelo, which we determined to be 1.8±0.1 kb/min. This value aligns with previous measurements reported in the literature [Our kernel-based deconvolution approach was validated by control measurements involving dual-color tagging of the gene body, both at 5’ and 3’ regions . These mterature , 30 . Consequently, these rates are independent of gene length, allowing for direct comparisons across different genes. This facilitated the intriguing observation that the genes reach a similar maximum average transcription rate, denoted as R = 〈r〉 . Moreove= 〈r〉  of single-allele transcription rates r that yield a similar mean transcription rate R, an intriguing pattern emerges. These distributions converge consistently across different genes , can be decomposed into two distinct parameters: the instantaneous probability of an allele being in the ON state (PON(t)), representing the fraction of ON alleles at time t, and the mean initiation rate (K(t)) for ON alleles. Given the above decomposition, most of the variation in R could arise from changes in either K, PON, or both.The next goal of our analysis is to elucidate how the consecutive switches between ON and OFF periods quantitatively govern the transcription rate hb, we estimate the time-dependent parameters R(t) and PON(t) for each AP bin. To obtain R(t), we calculate the average of ~250 single-allele instantaneous transcription rates (PON(t) by quantifying the fraction of alleles in the ON state at each time point (K(t), we average initiation events restricted to the ON state. By repeating this procedure for all AP positions, we reveal the spatiotemporal variations in PON and K , we obtain distinct spatiotemporal PON profiles  aligning quantitatively with the time scale predicted by the auto-correlation analysis above  accentuates the limited subset of the parameter space utilized and underscores the stringent quantitative relationships emerging from our dataset  tend to achieve higher expression levels mainly by reducing TOFF, medium-to-high-transcribing alleles are predominantly tuned by extending TON (F = 1/(TON + TOFF)) govern tuning of the transcriptional activity of low-transcribing alleles, while changes in burst size (B = K · TON) exert greater influence on the tuning of medium-to-high-transcribing alleles . HoweveTON and TOFF account for the change in transcriptional activity following a perturbation. In essence, since these relationships relate any given PON to specific TON and TOFF pairs, a measured change in PON upon a perturbation provides a prediction as to which of the two parameters was primarily altered. Remarkably, for each type of the examined perturbations, we find both predominant TON and TOFF modulation at different spatiotemporal coordinates  might not be inherent to the examined regulatory manipulation , but rather a consequence of the expression range (the PON regime) of these genes.To further explore the generality of these observations, we examined data from two previous studies in the early fly embryo. In one study, transcription was modulated by varying BMP signal, a dorsoventral morphogen , while iDrosophila embryo. The wide range of transcriptional activities that shape protein abundance in this system played a crucial role in our ability to uncover the fundamental relationships governing bursting dynamics. Rather than specific regulatory processes being inherently tied to distinct bursting parameters, our data suggests that most transcriptional tuning is channeled through a single control parameter: the ON-probability PON (TC) of approximately a minute. This time constant explicitly links the mean durations of ON and OFF periods, which are thus predominantly determined by the control parameter, PON. Therefore, gene activity, as characterized by PON (given a relatively constant initiation rate), conveys extensive information about the underlying bursting parameters.In this study, we developed an approach to quantify real-time single allele transcriptional bursting in the context of the developing early lity PON . MoreoveAs our parameter estimation does not rely on fitting a specific mechanistic model of transcription to the data but, instead, directly identifies ON and OFF periods from single-allele initiation events, the uncovered bursting relationships are both effective and broadly applicable across various models. Any detailed N-state model , 21, 33 TON and TOFF — or, equivalently, the constancy of TC — is not inherent to prevailing models of transcription and isn’t readily mapped to commonly invoked molecular processes. This strongly suggests an as-yet-unappreciated mechanistic constraint. Given the conserved nature of the transcription machinery and regulatory mechanisms across eukaryotes, it is highly probable that such a fundamental constraint, and the identified relationships more broadly, will apply to a wider range of systems [Notably, the linkage between  systems .K, may differ [PON < 0.5) [Comparing quantitative parameters between systems is challenging due to measurement and analysis differences across distinct experimental setups. However, we managed to examine bursting parameters derived from extensive perturbations of a yeast gene , and we y differ , 27  .PON. Of even greater intrigue is deciphering how the constancy of TC is molecularly implemented. Our work suggests a mechanism that operates independently of the specific gene locus and might be conserved across eukaryotic systems. Intriguing possibilities involve the transcriptional environment, including structural aspects of nuclear architecture or the molecular assembly and disassembly of transcription machinery components and their localized accumulation [The discovered bursting relationships, and their potential applicability to other species, warrant the development of a novel mechanistic framework for transcriptional regulation. In future studies, it will be imperative to unravel the molecular mechanisms responsible for integrating diverse regulatory processes  into the control parameter mulation –52.TC, could play a key role in transcription noise filtering and efficient regulation. Its measured small value minimizes noise, as bursts are easily buffered by longer mRNA lifetimes. Moreover, the small TC allows gene transcription to respond rapidly to input TF changes (In addition to opening new research directions into the molecular underpinning of the observed relationships and conserved parameters, our study raises questions about the functional consequences and potential benefits of this identified regime. For instance, the central time scale of the bursting dynamics,  changes –D. This 1