Csn1s2b locus. Through experimental mouse genetics, we dissect the lactation-specific distal enhancer bound by the mammary-enriched transcription factors STAT5 and NFIB and the glucocorticoid receptor. While deletions of canonical binding motifs for NFIB and STAT5, individually or combined, have a limited biological impact, a non-canonical STAT5 site is essential for enhancer activity during lactation. In contrast, the intronic enhancer contributes to gene expression only in late pregnancy and early lactation, possibly by interacting with the distal enhancer. A downstream super-enhancer, which physically interacts with the distal enhancer, is required for the functional establishment of the Csn1s2b promoter and gene activation. Lastly, NFIB binding in the promoter region fine-tunes Csn1s2b expression. Our study provides comprehensive insight into the anatomy and biology of regulatory elements that employ the JAK/STAT signaling pathway and preferentially activate gene expression during lactation.Enhancers are transcription factor platforms that synergize with promoters to control gene expression. Here, we investigate enhancers that activate gene expression several hundred-fold exclusively in the lactating mouse mammary gland. Using ChIP-seq for activating histone marks and transcription factors, we identify two candidate enhancers and one super-enhancer in the  Csn1s2b locus during lactation.Enhancers and promoters work together to actively regulate gene expression affecting several biological processes. Here, the authors provide molecular insights into the regulation of enhancers and super-enhancers in the  While current approaches, such as the ChIP-seq and physical contact studies, are useful in identifying candidate enhancers, their biological predictions are limited and validation through genetic experiments is needed. Enhancers are occupied by multiple transcription factors (TFs) that might bind directly to DNA through their respective recognition motifs or indirectly through tethering. Since experimental genetic studies generally ablate the entire enhancer, the structural and functional contribution of individual TFs remains to be understood.Enhancers are transcription component platforms that control the location, timing and intensity of gene expression5. STAT5 is activated by prolactin and it controls mammary alveolar development during pregnancy and the activation of genetic programs resulting in lactation4. While most STAT5 target genes are highly induced during pregnancy and to a lesser extent during lactation6, the activation of the Csn1s2b gene7 occurs preferentially during lactation8, possibly through enhancers that are specifically established after parturition8. ChIP-seq profiles for STAT5 and H3K27ac and other mammary-enriched TFs suggested the presence of highly complex mammary enhancers8. Although most of these enhancers appear to depend on STAT5 as the anchor for the establishment of larger protein complexes, the stage-specific generation of enhancers remains to be understood. It is not known why seemingly structurally identical enhancers can be activated by pregnancy hormones either during pregnancy or lactation.Several hundred genes are uniquely expressed in mammary tissue and activated by pregnancy and lactation hormones through the tyrosine kinase JAK2 and the transcription factors Signal Transducer and Activator of Transcription (STAT) 5A and 5B (referred to as STAT5)ODAM) gene being possibly a founding member. While a CSN3-like protein is already found in early amniotes and appears to be the first member of the family of five caseins, CSN1s2b is a more recent addition that evolved through gene duplication9. The mouse casein locus spans ~400 kbp and consists of five casein genes  and at least three SCPP genes  that are expressed in salivary glands and possibly other secretory tissues. The casein locus remains a fertile ground for exploring tissue-restricted and hormone-controlled gene regulation. Foremost, while the casein genes are expressed exclusively in mammary tissue and are induced by pregnancy and lactation hormones, the interspersed genes are expressed preferentially in salivary gland tissue. Among the five casein genes, expression of Csn1s2b is uniquely different from the other four in that its activation predominantly occurs during lactation and not during pregnancy. The evolution of the five caseins through gene duplication begs the question to what extent regulatory elements were duplicated, developed de novo or even shared between genes. It seems plausible that regulatory elements controlling the ancient SCCP genes were repurposed and acquired features that permitted their activity in secreting mammary gland cells. It also remains to be determined whether regulatory elements controlling the ancestral Csn3 gene were acquired by the younger Csn1s2b gene, which is separated from Csn3 by three SCCP genes.Caseins, the major components of milk, are cardinal proteins that are unique to mammals. They are evolved from secretory calcium-binding phosphoproteins (SCPP) with the odontogenic ameloblast–associated  in the establishment of lactation-specific regulatory elements. For this, we employed experimental mouse genetics and functionally dissected the two enhancers, the super-enhancer and the Csn1s2b promoter. This permitted us to define the contributions of individual enhancers and the significance of STAT5 and NFIB in the activating the Csn1s2b gene during pregnancy and lactation.Here, we used ChIP-seq for activating histone marks and transcription factors to identify candidate enhancers in mammary tissue during pregnancy and lactation. We identified two candidate enhancers and one super-enhancer in the extended Csn1s2b is activated preferentially, and up to several-hundred-fold, during lactation, suggesting the presence of distinct regulatory elements. The adjacent Csn1s2a and Csn1s2b genes, which arose by gene-duplication prior to the split of eutherian mammals11, are subject to a different regulation. While Csn1s2b expression increased more than 250-fold between day 1 of lactation (L1) and day 10 (L10), Csn1s2a expression increased approximately 6-fold  and remained high throughout lactation, STAT5 binding at the candidate Csn1s2b enhancer was marginally detectable at p18 and was fully established between L1 and L10  bound by STAT5 and other TFs, including the glucocorticoid receptor (GR), NFIB and MED1, was identified 2.3 kb 5’ of the TSS Fig. . STAT5 aene Fig. . While uCsn1s2b DE do not overlap  was confirmed by the raw read mapping  were reduced by ~89%   and a 36 bp deletion covered the GAS and NFIB motifs (ΔIE)  Fig. . Residuarol Fig. . Deletioely Fig. . In contL10 Fig. .Fig. 4ThCsn1s2b expression throughout lactation and the intronic enhancer is most prominent at the intersection of pregnancy and early lactation. While there are no additional overt enhancer marks between the body of the Csn1s2b gene and the neighboring Csn1s2a and Prr27 genes, we explored the possibility of further enhancers and monitored activating chromatin marks in the extended casein locus at L1  and intronic enhancers (IE) is reflective of loss of gene expression. Notably, deletion of the SE severely impacted STAT5 binding at the Csn1s2b promoter but less so at the DE .Deletion of the SE resulted in the complete loss of H3K27c marks and transcription factor binding at that region Fig. . Importa DE Fig. . ConversCsn1s2b expression was reduced by ~60% 16. Within mammary enhancers the percentage is even higher. It remains to be determined whether STAT5B recognizes the canonical GAS motif in mammary tissue. In liver STATB levels exceed STAT5A by approximately 10-fold and the majority of STAT5B ChIP-seq peaks coincide with the classical GAS motif24. A similar observation was made in T cells20. While GAS motifs with a 4 bp spacer are generally recognized by STAT6, another STAT member contributing to the differentiation of mammary alveolar cells25, this non-canonical site is also recognized by STAT5 in the Csn1s2b distal enhancer.Unlike other genetically validated enhancers where STAT5 binds to a canonical GAS motifCsn1s2b26, can bind to the enhancer lacking the DNA binding motif adds further intrigue and suggests that the recruitment of multiple TFs can be facilitated through a single anchor, STAT5 in mammary enhancers. Our results also suggest that GR binding to its half-site within the distal enhancer might require the cooperative presence of a neighboring STAT5 site. Alternatively, GR could tether to STAT5 as shown in the Wap gene super-enhancer that contains a STAT5 binding motif but lacks a GR motif19. The progesterone receptor (PR) binds to GR motifs27 and is required for mammary alveolar development29. We analyzed PR ChIP-seq data from mammary tissue from progesterone treated non-parous mice30 and no binding was detected at the Csn1s2b distal enhancer with a PR half-site motif. However, conclusions from these experiments are limited since they were conducted in non-parous mice that lack the differentiated alveolar compartment. Similarly, ChIP-seq data from the estrogen receptor (ER) did not reveal any binding31. It is conceivable that the presence of four TF binding motifs in the distal enhancer region permits additional TFs to bind through less conserved DNA binding sites. The presence of four TF binding blocks in the distal enhancer, possibly in synergy with additional promoter and intronic elements, enables high Csn1s2b expression levels during lactation. It remains to be elucidated why overtly equivalent enhancers activate other casein genes already during pregnancy. Of note, the more than 95% reduction of Csn1s2b expression caused by the deletion of the distal enhancer had no overt impact on lactation and is in agreement with other species that lack a functional Csn1s2b gene.Our finding that NFIB, a critical co-activator for a range of mammary genes, including 16 and their contributions in activating these genes during pregnancy and lactation have been investigated in mutant mice lacking these TFs. Since the global deletion of transcription factors can have widespread consequences on a given cell, such experiments may have limited impact on understanding their role on specific genes. As such, proliferation and differentiation of mammary epithelium during pregnancy is greatly impaired in the combined absence of Stat5a and Stat5b, thus making it impossible to define their gene-specific roles. Targeted deletions of three STAT5 sites, in the mouse Wap locus, individually or in combination, defined redundant and non-redundant functions of an enhancer structure essential for gene activation during pregnancy19. In a classical study, Burdon and colleagues32 employed transgenic mice to explore three STAT5 binding sites in a sheep β-lactoglobulin transgene32 and suggested some degree of cooperativity between canonical and non-canonical STAT5 binding sites. However, not all genes under the control of STAT5 rely on cooperativity22. A role for the GR in mammary gene regulation is not that clear. Mice lacking the GR33 or expressing a GR devoid of its DNA binding domain34 displayed a slightly impaired development of mammary ducts in non-parous mice. However, alveolar differentiation during pregnancy and expression of milk proteins during lactation were unimpaired, demonstrating that the GR is not required for normal mammary function and a potential compensatory function of the mineralocorticoid receptor has been proposed33. In contrast to the GR, the presence of the PR is required for the outgrowth and branching of mammary ducts35. However, since mice lacking the PR are infertile, mammary development and function during pregnancy has not been investigated. Moreover, PR ChIP-seq data from lactating mammary tissue are not available. However, studies using mouse mammary cell lines have revealed important functions of the PR in the regulation of the Csn2 gene36. NFIB isoforms are abundantly expressed in mammary tissue37 and deletion of the Nfib gene by itself or in combination with Stat5 supported the concept of cooperativity in gene activation in mammary tissue26.STAT5, GR and NFIB jointly occupy candidate enhancers of ‘mammary-specific’ genesCsn1s2b. We propose that this SE, which is located next to the Odam gene and separated from Csn1s2b by the Prr27 gene, was part of the evolutionary older Csn3 gene, and the younger Csn1s2b gene captured its activity. Csn3 and its neighboring odontogenic ameloblast–associated (Odam) gene originated from a common precursor9 and the gene arrangement in this locus  predates the emergence of Csn1s2b. In contrast to Csn1s2b, pregnancy expression of the neighboring Csn1s2a gene is not overtly controlled by the SE. The molecular mechanism underlying lactation failure in mice lacking the SE needs further investigation and might be the result of deregulation of the entire locus. Two CTCF binding sites associated with this SE38 might be an early signature of the casein locus before its expansion that added four additional casein genes, including Csn1s2b. However, deletion of these CTCF binding sites, did not alter Csn1s2b expression38, suggesting that they have limited biological activity.In addition to the distal enhancer, we identified a super-enhancer (SE) essential for the activation of the 43. However, as stated by one of the reviewers, we humans are not always good in selecting the TF sites or potential regulators that turn out to be important or essential. As shown here, uncovering the function and complexity of enhancers requires detailed genetic interventions. The casein locus with five genes expressed exclusively in mammary glands, three interspersed genes expressed in salivary glands and at least 20 candidate mammary enhancers, super-enhancers and CTCF sites remains a case study in evolutionary strategies to ensure uncompromised gene regulation. As further genetic inquiries are conducted, the multiplicity of regulatory building blocks controlling mammary- and salivary-gland specificity and cytokine-induced gene activation will continue to unfold.The increasing use of a wide range of ChIP-seq and chromatin capture approaches suggests that the mammalian genome is riddled with candidate enhancers that potentially control the spatio-temporal expression of lineage-specific genesCas9 mRNA were co-microinjected into the cytoplasm of fertilized eggs for founder mouse production. The ∆N/S2 and ∆S2/3 mutant mouse was generated by injecting sgRNAs for NFIB site into zygotes collected from ΔS2 mutant mice. All mice were genotyped by PCR amplification and Sanger sequencing (Macrogen and Quintara Biosciences) with genomic DNA from mouse tails  and all animal experiments were approved by the Animal Care and Use Committee (ACUC) of National Institute of Diabetes and Digestive and Kidney Diseases  and performed under the NIDDK animal protocol K089-LGP-17. CRISPR/Cas9 targeted mice were generated using C57BL/6 N mice (Charles River) by the transgenic core of the National Heart, Lung, and Blood Institute (NHLBI). Single-guide RNAs (sgRNA) were obtained from either OriGene  or Thermo Fisher Scientific , GR , NFIB , H3K27ac , RNA polymerase II , H3K4me1  and H3K4me3 . Libraries for next-generation sequencing were prepared and sequenced with a HiSeq 2500 or 3000 instrument (Illumina).Mammary tissues from specific stages during pregnancy and lactation were harvested, and stored at −80 °C. The frozen-stored tissues were ground into powder in liquid nitrogen. Chromatin was fixed with formaldehyde  for 15 min at room temperature, and then quenched with glycine . Samples were processed as previously describedhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality filtering and alignment of the raw reads was done using Trimmomatic44 (version 0.36), Bowtie45 (version 1.2.2) and Samtools46 (version 1.8), with the parameter ‘-m 1’ to keep only uniquely mapped reads, using the reference genome mm10. Picard tools  was used to remove duplicates. Homer47 (version 4.8.2) and DeepTools48 (version 3.1.3) software was applied to generate bedGraph files, separately. Integrative Genomics Viewer49 (version 2.5.3) was used for visualization. Each ChIP-seq experiment was conducted for more than two replicates. DeepTools was used to obtain the Pearson and Spearman correlation between the replicates.The raw data were subjected to QC analyses using the FastQC tool (version 0.11.9) (50 (version 2.2.7.1) peak finding algorithm was used. Peak calling of TFs and histone markers for WT and mutants was done for replicates, which were subsequently overlapped using Bedtools51 (version 2.29.2) to identify high-confident peaks. TF bound enhancers were considered as true enhancer elements if they showed H3K27ac underneath. Coverage plots  and motif analysis with default settings were done using Homer software.In order to identify regions of ChIP-seq enrichment over the background, MACShttps://CRAN.R-project.org/package=dplyr) and ggplot252 were used for visualization. Sequence read numbers were calculated using Samtools software with sorted bam files.Coverage plots were generated using Homer software with the bedGraph as input. R and the packages dplyr  on the CFX384 Real-Time PCR Detection System (Bio-Rad) according to the manufacturer’s instructions. PCR conditions were 95 °C for 30 s, 95 °C for 15 s, and 60 °C for 30 s for 40 cycles. All reactions were done in triplicate and normalized to the housekeeping gene Gapdh. Relative differences in PCR results were calculated using the comparative cycle threshold (CT) method.Total RNA was extracted from frozen mammary tissue of wild type and mutant mice using a homogenizer and the PureLink RNA Mini kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Total RNA (1 μg) was reverse transcribed for 50 min at 50 °C using 50 μM oligo dT and 2 μl of SuperScript III (Thermo Fisher Scientific) in a 20 μl reaction. Quantitative real-time PCR (qRT-PCR) was performed using TaqMan probes . Ribosomal RNA was removed from 1 μg of total RNAs and cDNA was synthesized using SuperScript III (Invitrogen). Libraries for sequencing were prepared according to the manufacturer’s instructions with TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold  and 50 bp paired-end sequencing was done with a HiSeq 2500 instrument (Illumina).https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Total RNA-seq read quality control was done using Trimmomatic44 (version 0.36) and STAR RNA-seq53 (version 2.5.4a) using 50 bp paired-end mode was used to align the reads (mm10). HTSeq54 (version 0.9.1) was to retrieve the raw counts and subsequently, R (version 3.6.3) (https://www.R-project.org/), Bioconductor (version 3.10)55 and DESeq252 were used. Additionally, the RUVSeq56 package was applied to remove confounding factors. The data were pre-filtered keeping only those genes, which have at least ten reads in total. The visualization was done using dplyr (https://CRAN.R-project.org/package=dplyr) and ggplot257.The raw data were subjected to QC analyses using the FastQC tool (version 0.11.9)  on the CFX384 Real-Time PCR Detection System (Bio-Rad). The primers used were SE 5′-GTACTCTGGAAAAGTAGGCAGTGC-3′, Csn1s2b-DE 5′-AGCTGGCCAACACAAAAGAATGGC-3′, Csn1s2b-IE 5′- AGCCAGGTGAGTGAGCTATGTTC-3′, Csn3-E1 5′- GAGTCTAACCACGCTACAGCTTC-3′, and Csn3-E2 5′- GTAGCTACTTCGGAAACCATCAAGG-3′. Interaction frequencies were normalized to the values of an internal control.DNA samples for 4C-seq from our previous studyP < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001 was considered statistically significant. Significances for homer de novo motifs were evaluated with Poisson distribution59.For comparison of samples, data were presented as standard deviation in each group and were evaluated with a one-way ANOVA followed by Dunnett’s multiple comparisons test, 2-way ANOVA followed by Tukey’s multiple comparisons test for comparisons or unpaired two-tailed t-test between WT and mutants using GraphPad Prism 8 (version 8.2.0). A value of *Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


The eukaryotic genome is packaged into chromatin, a polymer of DNA and histone proteins that regulates gene expression and the spatial organization of nuclear content. The repetitive character of chromatin is diversified into rich layers of complexity that encompass DNA sequence, histone variants and post-translational modifications. Subtle molecular changes in these variables can often lead to global chromatin rearrangements that dictate entire gene programs with far reaching implications for development and disease. Decades of structural biology advances have revealed the complex relationship between chromatin structure, dynamics, interactions, and gene expression. Here, we focus on the emerging contributions of magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS NMR), a relative newcomer on the chromatin structural biology stage. Unique among structural biology techniques, MAS NMR is ideally suited to provide atomic level information regarding both the rigid and dynamic components of this complex and heterogenous biological polymer. In this review, we highlight the advantages MAS NMR can offer to chromatin structural biologists, discuss sample preparation strategies for structural analysis, summarize recent MAS NMR studies of chromatin structure and dynamics, and close by discussing how MAS NMR can be combined with state-of-the-art chemical biology tools to reconstitute and dissect complex chromatin environments. In the nuclei of eukaryotic cells, approximately 2 m of DNA must be packaged and organized for efficient gene regulation and DNA replication . On a glBottom-up approaches using purified components are well suited to systematically probe the interplay between nucleosomes and chromatin structure on the Ångstrom and nanometer scale . The isoBuilding upon these studies, the interactions of many adjacent nucleosomes can be addressed. The chromatin context is important for biomolecular recognition; some chromatin modulator complexes are much larger than a nucleosome and can sense nearby nucleosomes , many arin vitro and in nucleo  achieves partial averaging by rapid (tens of kilohertz) rotation of the sample at 54.7° (the magic angle) relative to the external magnetic field . MAS then nucleo , and than nucleo . Therefon nucleo .in vitro biochemical and structural chromatin studies. Nucleosome arrays have been standardized to include twelve 601 sequence repeats, partitioned by selected lengths of linker DNA , arguing for a much more flexible landscape than suggested previously by X-ray crystallography and hydrogen-deuterium exchange experiments , for example, is a hydrophobic modification thought to increase the compaction of nucleosome arrays by altering the adjacent residue side-chain interactions with DNA . Shoaib 1H-detection and generated highly resolved spectra of the histone tail backbone. Crucially, the spectral quality provided residue-specific assignments to directly compare the isotropic chemical shifts with solution NMR experiments. PHD2 binding produced comparable 1H-15N resonance broadening of residues 3–10 in both conditions. While the weak affinity of PHD2 complicated quantitative comparison of binding interactions, this study confirmed the permeability of dense chromatin environments to histone readers. This strategy will undoubtedly prove useful in future experiments aimed to dissect the interactions between histone readers and their nucleosome binding sites.MAS NMR has also revealed that histone tails can sense their local environment and drive chromatin compaction while remaining dynamic and potentially accessible to regulatory proteins. A recent study by le Paige et al. confirmed that the dynamic tails sustain accessibility within dense sedimented samples . In this15N-13C-13C three-dimensional experiments  that were diminished in the 13C and 15N dimensions. Importantly, the absence of peak-splitting suggests that the LANA peptide bound both sides of the nucleosome simultaneously. Such an observation is important because the LANA peptide affects chromatin compaction by shielding the acidic patch  in the presence and absence of nucleosome arrays . HP1α isparation , these uE. coli or after purification may produce homogeneously modified proteins. For example, we have used co-expression with the kinase CK2 to prepare isotopically labeled and well-defined phosphorylated HP1α samples with high yields  . In thislabeling . This selabeling . It is alabeling . The unplabeling . While Nlabeling .E. coli cultures are then transformed with the tRNA/tRNA synthetase plasmid and a plasmid that contains the gene for the protein of interest with the TAG mutation. The unnatural amino acid is added to the media, and expression and purification of the modified protein can be performed as usual. While amber suppression systems have been engineered for the introduction of more than 100 UAAs  incorporation by genetic means (amber suppression)  Figure . Typical100 UAAs , the UAA100 UAAs . The maj100 UAAs .13C, 15N labeling and the other at natural abundance. Once the segments are purified, mixing of the segments results in an interaction between the intein pieces which adopt a functional intein fold. The intein mediates the formation of a native peptide bond between the two protein segments while excising itself in the process. This process requires a cysteine residue at the ligation junction. The result is a full-length protein with only a segment of the sequence visible by NMR, thereby simplifying the acquired spectra. This can aid assignment protocols  will continue to improve signal sensitivity and facilitate the resolution of chemical shift perturbations and chromatin interactions  and the detection and analysis of ractions . Since cractions . Chemica


Moreover, MLL2 plays a critical role in development and germ line deletions of Mll2 have been associated with early growth retardation, neural tube defects and apoptosis that leads to embryonic death. It has also been involved in the control of voluntary movement and the pathogenesis of early stage childhood dystonia. Additionally, tumor-promoting functions of MLL2 have been detected in several cancer types, including colorectal, hepatocellular, follicular cancer and gliomas. In this review, we discuss the main structural and functional aspects of the MLL2 methyltransferase with particular emphasis on transcriptional mechanisms, gene regulation and association with diseases.The Mixed Lineage Leukemia 2 (MLL2) protein, also known as KMT2B, belongs to the family of mammalian histone H3 lysine 4 (H3K4) methyltransferases. It is a large protein of 2715 amino acids, widely expressed in adult human tissues and a paralog of the MLL1 protein. MLL2 contains a characteristic C-terminal SET domain responsible for methyltransferase activity and forms a protein complex with WRAD , host cell factors 1/2 (HCF 1/2) and Menin. The MLL2 complex is responsible for H3K4 trimethylation (H3K4me3) on specific gene promoters and nearby  Chromatin remodeling is a key feature of gene regulation and activity, with histone modifications playing a primary role in the modulation of the chromatin landscape and gene expression. Among the most prominent histone modifications is the methylation of histone 3 (H3) lysine (K) residues, detected on gene enhancers and specific gene promoter regions. Mono- and di-methylation of H3K4 H3K4me1/me2) is mainly observed in enhancers whereas H3K4me3 is present on active gene promoters. Several protein lysine methyltransferases (PKMTs), including Mixed Lineage Leukemia 1-5 (MLL1-5/KMT2A-E), SET Domain-Containing 7 (SET7), SET and MYND Domain-Containing 3 (SMYD3), SET9 and PR/SET Domain 9 (PRDM9), are responsible for the transfer of methyl groups onto H3K4. The largest group of human lysine 4 (K4) HMKTs is the Mixed Lineage Leukemia (MLL/KMT2) protein family, named after the association with a subset of incurable acute leukemias of its founding member. All family members are characterized by a highly conserved catalytically active Su(var)3-9, Enhancer of zeste and Trithorax (SET) domain . T,8. T6,8]The CXXC domain composed of two zinc ions and four cysteine residues (Cys4), also known as the Zinc-finger (ZF)-CXXC domain, recognizes and binds to non-methylated CpG DNA, being critical for the association of MLL2 to chromatin . Both MLNext to the ZF-CXXC domain, MLL2 protein contains multiple PHD fingers, PHD1 to PHD4  which poTaspase 1 exhibit defects in cell proliferation and at the progression of the cell cycle, indicating the functional significance of the MLL2 cleavage [MLL2 and MLL1 can be cleaved by threonine aspartase 1 (Taspase 1) . Upon clcleavage .Additional DNA-binding motifs have been detected in MLL1 and MLL2 in the form of multiple HMG-like N-terminal AT hooks which enable binding to AT-rich DNA, discriminating their binding activities from the other MLL family members .In addition to the characteristic SET domain, MLL2 contains a CXXC domain followed by 4 PHD (PHD1-4), a single bromo domain  as well as a FYRN and FYRC domain (created using BioRender).All MLL family proteins contain a highly conserved SET domain at their C-terminus. They all form multi-protein complexes known as COMPASS (complex of proteins associated with Set1) and COMPASS-like complexes based on their homology with Drosophila Trr, Trx and dSet1 ,20. ThesThe WRAD module regulates the enzymatic active form of the complex, confers stability and enables recruitment to chromatin. It is composed of WDR5 , RbBP5 , ASH2L  and DPY30 (homolog of Sdc1) subunits which are critical for H3K4 methylation activity ,21,22,23Each COMPASS complex contains additional unique subunits on top of the main interacting proteins that enable their functional diversity . For MLLMeis 1, Hoxa9, CDKN1B and CDKN2C, which are required for MLL fusion protein-mediated leukemogenesis. The interaction of MLL1 and Menin forms a binding pocket for LEDGF which promotes transcriptional activation and is necessary for leukemogenesis. MLL2 shares the same interaction with Menin but not with LEDGF which is considered a unique coactivator of MLL1 complex activity. Apart of the nuclear member of the A-kinase anchoring protein family, AKAP95, MLL2 interacts only with very few interacting partners [Menin has been shown to be necessary for MLL1 target gene expression such as partners .For the MLL3 and MLL4 COMPASS, the unique subunits are PTIP-associated 1 (PA1), PAX transactivation domain-interacting protein (PTIP), nuclear receptor coactivator 6 (NcoA6) and the H3K27me3 demethylase ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX) ,27,28, wThe COMPASS complexes are responsible for mono-, di- and trimethylation of H3K4  and accoComplex recruitment is mediated by several mechanisms to specific gene loci, either through binding to histone modifications, specific transcription factors, cofactors and long noncoding RNAs (lncRNAs). Of interest, both the core subunits as well as the complex-specific ones can interact with transcription factors to recruit the complexes to specific gene loci. Among them, Menin has been demonstrated to interact with estrogen receptor-α (ERα) and enables MLL2 recruitment to the gene locus . Other tMLL1 has been shown to interact with transcription cofactors, including the lysine acetyltransferases MOZ, CBP and MOF, which mediate gene expression through H4K16 acetylation. Furthermore, MLL1, but not MLL2, has been demonstrated to bind to the PAF1 complex, serving as a bridge for RNA pol II, indicating a unique function of MLL1 . AnotherRecently, elegant single-particle cryo-electron microscopy (cryo-EM) studies have shed some light on the way MLL complexes recognize H3K4 within the nucleosome core particles (NCP).The MLL1 complex was shown to dock on the NCP through the RbBP5 and ASH2L proteins which interact both with the nucleosomal DNA and the H4 tail. This configuration enabled the catalytic SET domain of MLL1 to align at the nucleosomal dyad and facilitate the symmetric access of H3K4 substrate to the NCP .Additionally, there is evidence that the methylation of H3K4 can be induced by mono-ubiquitination of the histone H2B. A specific H2B mark on lysine 120 (H2BK120ub1) has been shown to disrupt chromatin compaction and allow the open chromatin structure. Cryo-EM studies of MLL1 and MLL3 have demonstrated their association with NCPs that contain H2BK120ub1 or unmodified H2BK120. The RBBP5 of MLL1 or MLL3 binds directly to H2B-conjugated ubiquitin. This interaction enables access to the H3 tail, which is required for H3K4 methylation. The differential organization of WDR5 and RBBP5 in MLL1 and MLL3 complexes accounts for their distinct enzymatic activities ,47.de novo H3K4me3 in ESCs since its knockdown caused a global reduction in H3K4me3 [Another recent structural study demonstrated that the activity of the MLL family members on the NCP requires DPY30 . It was  H3K4me3 .Altogether, the core subunits of each MLL complex, as well as the proteins containing IDRs, exert important biophysical properties in MLL complexes and modulate their activity in chromatin.Several studies using Chromatin Immunoprecipitation (ChIP) followed by NGS sequencing (ChIP-seq) have shown that MLL2 can establish narrow H3K4me3 peaks at regions proximal to active gene promoters as well as co-exist with H3K27me3 in bivalent genes of embryonic stem cells (ESC). The purification of a minimal catalytical MLL2 complex (MLL2C) has demonstrated a specific methyltransferase activity for H3K4 methylation (H3K4me1/me2/me3) on recombinant histone octamers and recombinant chromatin. The stimulatory effect of the MLL2-related H3K4me on transcription has been validated using a well-established chromatin-templated in vitro transcription system . SpecifiBy using specific antibodies recognizing two different epitopes in the C-terminal portion of MLL2  and the ChIP-seq technique, a large number of MLL2-binding regions were identified with 70% localized to promoters, 14% to gene bodies and 16% to intergenic regions. The high occupancy of MLL2 in promoters was consistent with previous studies indicating its activity in bivalent genes. A further analysis with the ab CT1 revealed that more than 6000 MLL2 binding sites were outside of TSS, located in the intergenic and gene body regions. Moreover, the same study reported that around 39% of them shared marks with active enhancers, including p300 and H3K27ac . InteresIn order to reveal the molecular mechanisms that underly MLL2 targeting to chromatin, the CRISP/Cas9 technology was used to generate MLL2 knockout mESC and was used for future rescue experiments. MLL2 was shown to bind unmethylated CpG-containing DNA indicating a possible involvement of the CXXC domain. Using several CXXC mutants, it was observed that MLL2 depends on its CXXC domain for recruitment to chromatin . ThroughKlf4, Oct4), but is rather necessary for the expression of master regulators required for the primordial germ cell (PGC) specification, such as Prdm1, 14, DDx4 and Lin28b, during ESC differentiation [Studies on MLL2 knockout mESC have demonstrated that MLL2 is not required for the expression of pluripotency genes , which revealed the concomitant interplay of H3K27me3, MLL2-dependent transcription regulation and DNA methylation. Genome-scale screening indicated that the depletion of CXXC1 (component of SET1A/B complexes) in MLL2 knockout mESCs was sufficient to rescue the loss of expression of the ~1200 MLL2-dependent genes. Interestingly, the rescue of these genes expression was not correlated with the re-appearance of the H3K4me3, showing that MLL2 and H3K4me3 may have a more instructional role in gene regulation for certain types of genes, as has been reported in previous studies [Of importance, a study showed that a large set of genes which exhibited increased levels of H3K27me3 upon MLL2 depletion, could be rescued by the removal of DNA methylation or the depletion of members of the PRC2 complex . This ov studies ,61.Additionally, in the vast majority of MLL2 targeted genes, the deletion of MLL2 led to the reduction in gene expression, while this repression was shown to be restored by either the removal of H3K27me3 or restoring the DNA demethylation. It was observed that there is a big overlap among genes at least partially rescued that could be explained from the fact that DNA methylation impacts H3K27me3 deposition . The remMll2-conditional knockout mammalian ESCs have revealed the order of the events that lead to gene silencing and the crosstalk between the action of MLL2, RNA pol II processing and DNA methylation. Focusing at the MagohB gene, they have uncovered the mechanistic role of MLL2 in gene expression. The presence of MLL2 maintained an open chromatin state at the promoter of the target gene, regulated RNA pol II association and was correlated with active chromatin marks and high levels of mRNA. The depletion of Mll2 led to a rapid decrease in active marks (H3K4me3 and H3K9Ac) and an increase in DNA methylation at the MagohB gene promoter. Interestingly, DNA methylation seemed to be a secondary event to gene silencing [Transcriptional kinetic studies in ilencing .To further evaluate the role of DNA methylation in antagonizing MLL2 in d25 GV oocytes, a study assessed the distribution of H3K4me3 in the absence of DNA methylation by using conditional knockout mice for MLL2 and double knockout for DNMT3a/b genes. It was revealed that there are two complementary, independent mechanisms of H3K4m3 trimethylation. One mechanism was transcription-dependent and was not connected to MLL2 activity, while the other relied on the specific targeting of MLL2 in unmethylated CpG-rich regions, mainly at distal elements and intergenic regions. Interestingly, these regions are protected by DNA methylation during oogenesis. The non-canonical role of MLL2 was not connected to gene expression but rather marked the bivalent chromatin state at repressed H3K27me3-marked promoters ,64.ASB2) which in previous studies had been demonstrated to be induced by retinoic acid in leukemia cells. ASB2 expression in myeloid leukemia cells has been shown to induce the inhibition of proliferation and chromatin condensation. However, −/−MLL2 cell lines have shown a reduction in ASB2 expression due to an effect on H3K4me3 levels [Additional studies on the epigenetic and expression profiling of target genes were performed to detect pathways that are regulated by MLL2 enzymatic activity and revealed mechanistic insights into the functional role of MLL2 . Upon Ch3 levels .The same genome-wide study demonstrated the involvement of MLL2 in different cellular pathways. Among the transcription factors that were regulated by MLL2 was the Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) and p53. It has been demonstrated that p53 contains a sequence similar to an autoinhibitory N-terminal loop of the MLL2-SET domain. The N-terminal loop of MLL2 adopts a similar conformation as the H3 tail and, thus, enters the substrate-binding pocket of another MLL2-SET enzyme. This specific sequence and conformation of p53 makes it a perfect candidate substrate of the complex . FurtherMLL2 gene was originally identified by its homology to MLL1 and was further detected to be broadly expressed in human tissues. The ability of both paralogs to bind Menin/LEDGF, has proved critical for their normal functions [The unctions ,67.Mll2 germ-line deletions have been associated with early growth retardation, neural tube defects and increased apoptosis that leads to embryonic death before E11.5 [Mll2 was involved in the preservation of the mesodermal marker Mox1 and Hoxb1 as well as in the deregulation of HoxB cluster genes. However, after E11.5, Mll2 loss was not associated with notable pathologies indicating that it is not required for the late development and homeostasis of somatic or stem cells [Of importance, re E11.5 . Mll2 waem cells .Mll2 resulted in anovulation and death. The elevated transcription of apoptotic factors and p53 as well as the loss of global H3K4me2/3 was also detected [Mll2 deletion did not affect the global methylation of H3K4 and hematopoiesis, as observed with Mll1 [However, MLL2 is also implicated in germinal cell differentiation and contributes to enriched H3K4me3 marks observed in the active genes of spermatogonial stem cells. Spermatogenesis was lost upon its deletion . Moreovedetected . Additioith Mll1 .MLL2 for proper cytokine signaling. Upon stimulation by lipopolysaccharide (LPS), a Rosa-CreERT2 model of Mll2−/− macrophages from bone marrow, displayed attenuated intracellular NF-κB signaling due to reduced Toll receptor 4 (TLR4) activation. This was attributed to the loss of Phosphatidylinositol Glycan Anchor Biosynthesis Class P protein (Pigp) which adds glycophosphatidylinositol to transmembrane proteins. In turn, this induced the loss of CD14 anchoring at the cellular membrane which co-operates with TLR4 in response to LPS. Apart of the Pigp gene promoter, several other Mll2 targets exhibited reduced H3K4me3 peaks in TSS and a respective increase in H3K27me3 mark which relate to repressed or bivalent genes. Therefore, MLL2 possibly maintains the expression of the target genes through H3K4me3 promoter enrichment and the resistance of invading repression complexes. However, H3K4 hypomethylated genes in Mll2−/− macrophages exhibited no change in expression levels, indicating a higher sensitivity of some genes to H3K4me3 promoter depletion than others [Although the majority of hematopoietic cell types do not depend on MLL2 for their function, macrophages have been demonstrated to require n others ,72.MYC oncogene. MLL2 is attracted to the MYC enhancer by a process that involves β-catenin and promotes the transcription of MYC via H3K4me3 methylation [Furthermore, MLL2 is involved in cell growth control by regulating the activity of the hylation .MLL genes have been detected in several developmental disorders as well as in hematological and non-hematological cancers.The regulation of transcription by MLL family members is very important for human health, and mutations in MLL2 gene and characteristic brain magnetic resonance imaging findings with a typical facial appearance and possible progress to cranial and laryngeal dystonia over time [An important physiological role of MLL2 has been demonstrated in the control of voluntary movement. Specifically, MLL2 haploinsufficiency has been linked to the most severe type of a hyperkinetic movement disorder, the early onset-generalized children dystonia, which is defined by involuntary twisting postures due to sustained or intermittent contractions of agonist and antagonist muscles ,74,75. Tver time .MLL2 mutations has shown that certain proteins associated with dystonia, such as torsin family 1 member A (TOR1A), THAP domain-containing, apoptosis-associated protein 1 (THAP1) and dopamine receptor D2 (D2R) are decreased in cerebrospinal fluid and fibroblasts, indicating MLL2 implication in disease pathogenesis that needs further investigation [Gene expression profiling in patients harboring tigation .MLL2 deletion in excitatory forebrain neurons resulted in learning impairment due to increased activity of genes involved in hippocampal plasticity via H3K4me2/3 [Moreover, in adult mice, conditional 3K4me2/3 ,76.MLL1 have been associated with cancer onset. The MLL1 gene exhibits a considerable number of rearrangements with several other translocation partner genes, possibly attributed to the inability of developing hematopoietic cells to repair the frequent chromosomal double-strand DNA breaks [MLL1 to its fusion partners, the H3K4 methyltransferase activity is lost due to the loss of the SET domain. More than 135 MLL1 rearrangements have been identified up to date, being mostly in frame translocations that lead to the generation of gain-of-function oncoproteins with altered activities [Another disease-promoting role has been attributed to MLL2 in respect to cell proliferation enhancement and carcinogenesis . As origA breaks . The MLLtivities . The fusMLL1, including MLL-ENL, MLL-ELL, MLL-AF4, MLL-AF9, MLL-AF10 and MLL-PTD, accounting for 80–90% of MLLs, whereas MLL1-rearranged leukemias account for 10% of all leukemias [It is interesting to note that although MLL2 exhibits a structural similarity to MLL1, it is not related to chromosomal translocations and exhibits a lower affinity for DNA binding at unmethylated CpG sequences, being unable to replace MLL1 in leukemic oncoproteins ,77,78. Seukemias ,77.MLL2 gene (wt) was shown capable of decreasing the leukemic cell survival, but WT-MLL1 deletion had no impact on leukemia cell function, since targeting the N-terminal part, that is shared in the MLL1-fusion protein, did ablate leukemia cells [In MLL1-rearranged leukemias (MLL-AF9), deletion of the ia cells ; thus, iia cells ,80.Mll2 mutations in mice were not capable of inducing carcinogenesis [MLL2 mutations detected in cancers are mostly nonsense, missense or frameshift, and mainly involve the PHD and SET domains [MLL2 have been detected in neurofibromatosis 1-glioblastoma (NF1-GBM), leading to the truncation of the MLL2 protein and have been associated with early steps of gliomagenesis [Of interest, conditional or germline ogenesis ,68. Howe domains ,82,83. Aagenesis .The overexpression of MLL2 has also been detected in pancreatic cancer cells and additional translocations have been observed in glioblastomas .c-MYC, inducing its transcription [ASB2 which was previously induced in leukemia cells. Other transcription factors that were regulated by MLL2 include NR3C1 and p53, explaining the potential mechanistic implication of MLL2 in cancer progression [In colorectal cancer, MLL2 has been reported to promote cell proliferation through physical interaction with β-catenin which allows the recruitment of MLL2 to the enhancer element of cription . MLL2 tagression .Additionally, MLL2 has been revealed in genomic studies as a recurrent target for the integration on oncogenic viruses (hepatitis B virus and adeno-associated virus type 2) of hepatocellular carcinoma (HCC) tissues ,87, indiMLL2 mutations were frequently detected at a similar rate to t translocation which is the molecular hallmark of the disease, indicating a central role of MLL2 in tumorigenesis [Furthermore, in follicular lymphoma (FL), igenesis .MLL2 were frequently detected at a 17.9% mutation rate [Finally, in squamous-cell cancer of the head and neck (SCCHN), somatic mutations of ion rate . Since tTaken together, all the significant progress that has occurred in recent years in understanding chromatin accessibility mechanisms and their role in gene regulation, H3K4me1/me2/me3-enriched genomic regions were demonstrated to be of primary importance. Furthermore, structural and functional studies of the MLL methyltransferase family in mediating these histone marks in specific tissues have revealed unique, non-redundant functions despite the similarity between paralogs.MYC gene, switching-off MYC-dependent leukemia [MLL2 is particularly significant in mediating H3K4me3 in specific promoters of development-related genes, but is also required for H3K4me3 accumulation on bivalent promoters in ES cells. Moreover, an extensive range of H3K4 methylation-reader domains has been detected in many transcriptional coactivators demonstrating the direct stimulatory effects of the MLL complex-mediated H3K4 methylation on transcription. Therefore, studies determining the factors that enable the recruitment of MLL1/2 complexes to specific loci in the genome are highly demanded. MLL2 plays multiple and significant roles in the regulation of physiological voluntary movement; it is involved in childhood dystonia and in the pathogenesis of several malignancies. It is, thus, important to determine how to target MLL2 with small molecule inhibitors in different settings. Current efforts are directed to the development of inhibitors that target H3K4 methyltransferase activities or MLL1/2-associated subunit interactions in controlling the H3K4 methyltransferase function of MLL complexes. Major efforts were focused on the identification of chemicals that treat leukemias caused by MLL1 rearrangements. Two molecules are currently in phase I/II clinical trials  for Menin–MLL inhibition  for MLL-rearranged leukemias which show promising results. Selected inhibitors can either act on proteins recruited to the MLL1 complex that are required to maintain the leukemic state or block the methyltransferase activity of MLL1 by interrupting its interaction with WDR5, Menin or LEDGF . Other aleukemia ,91. Impoleukemia .Furthermore, recent experimental evidence suggests that MLL-associated transcriptional regulatory mechanisms, independent of the H3K4 methyltransferase activities of the complexes, are also involved in gene regulation and need to be taken into consideration as well as further investigated in functional studies.


Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are good models for heterochromatin study. In S. pombe, H3K9 methylation and Swi6, an ortholog of mammalian HP1, lead to heterochromatin formation. However, S. cerevisiae does not have known epigenetic silencing markers and instead has Sir proteins to regulate silent chromatin formation. Although S. cerevisiae and S. pombe form and maintain heterochromatin via mechanisms that appear to be fundamentally different, they share important common features in the heterochromatin structural proteins. Heterochromatin loci are localized at the nuclear periphery by binding to perinuclear membrane proteins, thereby producing distinct heterochromatin foci, which sequester heterochromatin structural proteins. In this review, we discuss the nuclear peripheral anchoring of heterochromatin foci and its functional relevance to heterochromatin formation and maintenance.Budding yeast  Many DNA-templated processes, such as replication, transcription, and DNA repair, are regulated in the context of chromatin structure, which is classified into euchromatin and heterochromatin. Euchromatin is less condensed and more easily accessed by RNA polymerase II and transcription factors, which enables active transcription. In contrast, heterochromatin maintains highly condensed chromatin regions throughout the cell cycle, impeding the access of various transcription factors and causing gene silencing , 2. In hSaccharomyces cerevisiae and Schizosaccharomyces pombe are well-studied model systems for the investigation of heterochromatin. However, between the two model species, there are many differences in the mechanism of heterochromatin formation and gene silencing. Notably, although the methylation of histone H3K9 and HP1 are well-conserved in S. pombe, neither of this histone modification and heterochromatin factor exist in S. cerevisiae [S. cerevisiae, the formation and maintenance of heterochromatins are regulated by the silent information regulator (SIR) complex-silencing system [revisiae , 6. Instg system , 6, 7.S. cerevisiae and Swi6 in S. pombe, are sequestered in several foci at the nuclear periphery [S. cerevisiae, H3K9-methylated nucleosomes and Swi6 in S. pombe form phase-separated liquid condensates, which maintain distinct biochemical conditions distinguished from the outer environments [S. cerevisiae and S. pombe.Although the mechanisms for silent chromatin formation are different in both model systems, they share several essential common features. Heterochromatin regions and heterochromatin structural proteins, the Sir2/3/4 complex in eriphery –11. Afteeriphery –11. We weriphery , 12–14; eriphery , 12–14; ronments . Throughronments , 15. In ronments , 15, 16.S. cerevisiae, epigenetic silencing markers conserved in metazoans have not been identified. Specifically, there are no orthologous proteins of chromatin modifiers for silencing mechanisms, such as DNA cytosine methylation, H3K9 methylation, and H3K27 methylation, in S. cerevisiae. Instead, the silent chromatin structure in S. cerevisiae is maintained through the recruitment and spreading of the SIR complex, which is composed of Sir2, Sir3, and Sir4 [In and Sir4 .S. pombe, H3K9 methylation and Swi6 lead to heterochromatin formation. H3K9 methylation is a conserved histone modification, responsible for heterochromatin formation in a multitude of organisms from fission yeast to humans [S. pombe, but several H3K9 methyltransferases have been identified in mammalian cells, including SUV39H1/KMT1A, SUV39H2/KMT1B, SETDB1/KMT1E, dimeric `G9a/KMT1C-GLP (G9a-like protein)/KMT1D, and the PRDM family [Drosophila melanogaster retains Su(var)3–9, G9a and SETDB1 as the H3K9 methyltransferases [Arabidopsis thaliana, KYP, SUVH5, and SUVH6 are identified as H3K9 methyltransferases [Neurospora crassa, a filamentous fungus, the H3K9 methyltransferase is Dim5 [In M family . Drosophsferases . In Arabsferases . In Neur is Dim5 .S. cerevisiae, the SIR complex is recruited to silent loci by interaction with repressor proteins bound to heterochromatin-specific DNA elements, including silencers in silent mating loci and multiple Rap1 binding sites in telomeres [S. pombe, Swi6 is recruited to silent loci by binding to H3K9-methylated nucleosomes [In elomeres . In S. pleosomes , 21. Upoleosomes .S. cerevisiae, homothallic mating (HM) loci, telomeres, and rDNA loci are the silent chromatin regions [MAT locus—either “a” or “α”  [HM loci; these HM loci—HMRa and HMLα—are located at either side of the MAT locus and maintained in a silent state, called HM silencing S. cerevi   , autonomously replicating sequences (ARS) binding factor 1 (Abf1), and origin replication complex (ORC) [+-dependent histone deacetylase (HDAC) family, deacetylates acetylated H4K16 (H4K16ac) [Specific DNA elements in ex (ORC) , 26  . For exaex (ORC) , 30. AftH4K16ac) –34. DeacH4K16ac) –34. In tH4K16ac) . The SirH4K16ac) , 37. TheH4K16ac) . In rDNAH4K16ac) . Net1 isH4K16ac) , 39. TopH4K16ac) , 40, 41.H4K16ac) , 43. TheH4K16ac) .S. pombe are pericentromeric regions as well as telomeres and silent mating-type loci  and cnt  repeats. The cnt region is flanked by two imr sequences in an inverted orientation [cnt and two imr sequences, is flanked by otr (outer repeat) repeats [otr repeats contain dg and dh repeat sequences, which are the RNAi-dependent heterochromatin initiation sites [mat2P or mat3M—located at the MAT locus. Like S. cerevisiae, both mat2P and mat3M elements are maintained in a transcriptionally silent state  into centromeric chanisms –55  separated by a hinge region [Heterochromatin structure formation is dependent on the spreading of Clr4 and Swi6 , 52, 61.e region . These He region , 64. Howe region –67. On te region .IRC-L/R) regions that retain tRNA genes  [The SIR complex in affinity . Reconsty [−8 M) . The C-ty [−8 M) , 92, 93.y [−8 M) . Sir4 iny [−8 M) .Kd value in solution is less than 17 nM, which suggests that Swi6 exists as a dimer at the nanomolar scale [Heterochromatin structural proteins form oligomers , 14. Swiar scale , 64. Thrar scale . Howeverar scale . Furtherar scale .ura4 + reporter gene locus, and more Swi6 bound to the region in the Swi6-V82E Y131W substitution mutant [In a previous study, an in vitro binding assay between reconstituted mononucleosomes and Swi6 revealed that the strength of the interaction is proportional to Swi6 concentration . Specifin mutant .S. cerevisiae. Previous studies revealed that in vitro, two SIR complexes bound to three nucleosomes in a nucleosomal array [Oligomerization is also important for the assembly of the SIR complex in al array , 16. To al array , 16. Siral array , 96. Alsal array , 97.A Sir3 dimer forms bridges between neighboring nucleosomes in an unmodified nucleosome array, resulting in a compact chromatin fiber . PreviouS. pombe and the SIR complex in S. cerevisiae, is changed while binding to nucleosomes. A study suggested that Swi6 is altered to the spreading-competent form by binding to methylated H3K9 [The conformation of both heterochromatin structural proteins, Swi6 in ted H3K9 . In the ted H3K9 . Each ofted H3K9 . This auted H3K9 . Becauseted H3K9 , 14.S. cerevisiae, Sir2 is the NAD+-dependent histone deacetylase, which remove the acetyl group of the acetylated H4K16 residue [+ motif in the C-terminal of Sir3 [In  residue . During  of Sir3 , 29. The of Sir3 . Electro of Sir3 . Further of Sir3 .A recent study identified that the structure of H3K9-methylated nucleosomes is changed by binding to Swi6 . NotablyS. cerevisiae, the conformation of nucleosome structure is changed by the binding of the SIR complex or by the histone deacetylase activity of Sir2. A previous study performed analytical ultracentrifugation with nucleosome array assembled with recombinant histone proteins and DNA, and identified that formation of compacted chromatin fiber was suppressed by deletion of N-terminal stretch of histone H4 [In stone H4 . This restone H4 . Basic pstone H4 . H4K16 astone H4 . In addistone H4 . The deastone H4 .A study reported phase-separated liquid condensates in the presence of H3K9-methylated nucleosomes and Swi6 . Phase-sS. cerevisiae, whether SIR complex foci at the nuclear periphery are also the phase-separated liquid condensates remains to be identified.Notably, the shape of liquid condensates was similar to nuclear subcompartments filled with Swi6 proteins in the nucleus . DistincS. pombe and the SIR complex in S. cerevisiae, unless spreading is either prevented through heterochromatin boundaries or decreased gradually along the long transition zone in telomeres [Heterochromatin regions maintain a highly packaged chromatin structure throughout the cell cycle. Once heterochromatin is nucleated, heterochromatin is spread through the self-assembly of heterochromatin structural proteins, namely Swi6 in elomeres , 45.S. cerevisiae and S. pombe. In addition to S. cerevisiae and S. pombe, regional sequestration of heterochromatin structural proteins and heterochromatin loci is also conserved in mammals. The concept that the human nucleus consists of two megabase scale compartments, A and B compartments, was firstly suggested through previous Hi-C experiments [In this manuscript, we discussed the sequestered nuclear subcompartments of heterochromatin structural proteins and their role in heterochromatin formation in eriments . A compaeriments . Moreoveeriments . Integraeriments .Drosophila and mammals, the homologs of S. pombe Swi6, also forms phase-separated liquid condensates in vitro and in vivo, highlighting the importance of sequestered nuclear subcompartments of heterochromatin structural proteins and heterochromatin regions [S. cerevisiae and S. pombe, and even in mammals.The HP1α of  regions , 111. Si regions , 111. Th


Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression. The genome is a complex and very dense viscoelastic polymer matrix, and therefore, it is difficult to study individual components of the gene expression machinery with conventional light microscopy. The optical resolution of the conventional light microscopy is limited by the diffraction of light and allows to distinguish the objects only if they are ~200 nm apart. This ~200 nm resolution limit of the classical light microscope is given by the nature of light and results from the fundamental laws of physics. Ernst Abbe postulated in 1873 that the limit of discrimination will never pass significantly beyond half the wavelength of blue light [ibed DNA  or the uibed DNA  were posSuper-resolution microscopy (SRM), contrary to electron microscopy, allows for live-cell imaging at the previously unprecedented spatial and temporal level. Novel and progressive SRM approaches helped to uncover many previously undetected dynamic mechanisms that regulate mammalian RNA polymerase II (RNAPII) transcription. Nevertheless, RNAPII is not a solo player but its function is related to the overall gene expression processes and also intimately linked with the steps preceding as well as succeeding the transcription. In this review, we will introduce SRM techniques in general, we will put emphasis on single-molecule localization microscopy (SMLM), and present the variety of fluorophores suitable for SMLM. Then, we will first focus on SMLM applications to studying the dynamics of transcription factors, and finally on the RNAPII itself.Although Abbe postulated that the limit of discrimination is given by half the wavelength of blue light, he also acknowledged that this limit could be surpassed by applications based on principles that are outside of the law that he postulated ,2. FluorThe RESOLFT family ,9,13,14 SMLM, in contrast to the above-discussed deterministic SRM techniques, allows for the visualization of individual fluorophores at the level of individual molecules. This revolutionary approach is based on the temporal separation of individual fluorescent molecules based on a relatively simple principle that allows for the detection at subsequent time intervals of a sparse subset of well-separated fluorophores and reconstruction of a final super-resolved image from the localization of the centers of the fluorophores . SMLM tez-axis) is more challenging than in the imaging  plane for several reasons [z-axis utilize astigmatism [SMLM along the optical plane  illumination is commonly used in SMLM but suffers from significant drawbacks , such asSMLM and STED have recently met at their intersection called minimal photon emission fluxes (MINFLUX). This novel concept combines stochastic switching of the fluorophores on and off as in PALM or STORM but the emitter is localized by a doughnut-shaped excitation beam that is used in STED . As a reIn this review, we will further discuss the developments of the fluorescent labeling strategies for SMLM and then their use together with the above-reviewed SRM techniques to reveal the novel paradigms in the regulation and dynamics of RNAPII transcription.The rapid development of live-cell SMLM applications is linked to the progressive development of fluorophores with specific and unprecedented photophysical properties. Fluorophores used in SMLM can either be genetically encoded FPs, “self-labeling” tags such as HaloTag or SNAP that covalently associate with organic fluorochromes or organic fluorochromes such as Alexa Fluores attached directly to a primary antibody or attached to a secondary antibody directed against the primary antibody as in classical immunofluorescence protocols . A pleth2) intensity of 405 nm light allows for the photo-conversion of only a small subset of Dendra2 molecules in the sample. Favorable photo-physics [As mentioned above, individual fluorescent proteins are randomly photo-activated or photo-converted at different time points in PALM, while their neighbors remain dark or undetected ,25,30. T-physics  and low -physics  of Dendr-physics ,64,65,66-physics . However-physics ,68 that Genetically encoded self-labeling protein tags such as HaloTag and SNAP ,70,71 haThe development of PA fluorochromes extended the possibilities previously offered by PA- or PC-FPs. However, the dependence on the photoactivation for SMLM and SPT can be genetically circumvented by the precise copy number control of the fluorescently labeled fusion proteins . This apTaken together, the rapidly growing selection of highly specific fluorescent markers with superb properties binding to or being expressed as a part of individual molecules makes SMLM the method of choice to study the functional architecture of the cell nucleus and dynamics of transcription within its spatial context. Some of the key applications of fluorophores for SMLM studies of the RNAPII transcription and associated molecular processes are summarized in Diffusion of TFs allows them to search for their targets to initiate the transcription of specific genes and thereby regulate patterned gene expression ,82,83. ESeveral TFs were recently scrutinized by SMLM, which helped to uncover the common as well as distinct dynamic patterns, explaining the universal mechanisms as well as the roles of distinct TFs in distinct transcriptional programs. In general, the TF kinetics at the single-molecule level was inferred either by fast time-lapse imaging and SPT ,90 or bySPT in 2D of TMR-HaloTag-Sox2 visualized by epi- and BBSP illumination ,45 in liThe fact that only a small (3%) subpopulation of Sox2 molecules is bound to DNA at any given time  makes itThe single-molecule kinetics of steroid receptors, such as glucocorticoid receptor (GR) tagged with PC-FP mEos2 in living MCF-7 cell line was facilitated by RLS microscopy . The dif2/s and therefore the localization of individual TF molecules deviates from the classical Gaussian PSF. Izeddin et al. devised an algorithm that allowed continuous tracking of TFs diffusing at rates exceeding 10 µm2/s. This analysis revealed that although the dynamics of both TFs were similarly restrained by their interaction with nuclear structures, c-Myc and P-TEFb adopt different strategies for the exploration of nuclear space. While Dendra2-tagged c-Myc globally explores the nucleus and diffuses similarly to free Dendra2, individual Dendra2-tagged P-TEFb molecules explored the nuclear volume by sampling space of reduced dimensionality and displaying the characteristic of exploration constrained by fractal structures [Kinetic studies of yet other TFs, protooncogene c-Myc, and elongation factor P-TEFb revealed that despite their similar diffusion rates, each TF explores the space differently, highlighting their distinct roles in the regulation of transcription . SPT andructures .The discovery of the hierarchical and dynamic assembly mechanism of the general TFs suggests that this previously undetected mechanism of RNAPII pre-initiation is universal . In vitrTaken together, the single-molecule imaging uncovered previously unknown Sox2 EnCs positioning throughout the 3D space inside the nucleus and the likelihood of Sox2 finding and binding to EnCs. These detailed quantitative analyses of the Sox2 dynamics and spatial distribution of Sox2 EnCs provided valuable insight into the spatial organization and dynamics of the enhancer-binding pluripotency regulators in embryonic stem cells, which greatly extended our mechanistic understanding of the spatial and temporal regulation of gene expression in pluripotent cells and mammalian development at a molecular level. Moreover, the exemplar studies reviewed above underscored the advantages of single-molecule imaging and revealed previously undetected hierarchical transient-to-stable transition of TF-binding dynamics that regulate the assembly of RNAPII pre-initiation complex. In the next section, we discuss the recent progress in the understanding of RNAPII transcription dynamics with the examples of several detailed single-molecule imaging studies.S. cerevisiae, resulted in the higher RPB1 cluster dynamics and artificial lengthening of RPB1 CTD led to the lower dynamics of RPB1 clusters and their increased association with chromatin [Transcription by RNAPII is essential for gene expression and therefore fundamental for cellular activities. RPB1 enzyme, the large catalytic subunit of RNAPII central to the transcription process, forms spatially compartmentalized foci ,101,102.hromatin . Intriguhromatin , which ihromatin . Importahromatin  showed thromatin . The nothromatin . Anotherhromatin  that allhromatin . Therefohromatin .Spatial relationship and inferred functional correlation of RNAPII distribution and Sox2 enhancers was investigated in living mECSs by 2D SPT and mapping of Sox2 EnCs (as described in the previous section) by low-excitation and long-acquisition detection of stable DNA bound JF646-HaloTag-Sox2 molecules followed by PALM imaging of photoconverted Dendra2-RPB1 ,92. AutoThe dynamic clustering of RPB1 before productive transcription elongation revealed by tcPALM and SPT analyses has implications for the fine regulation of gene expression and was further confirmed by detailed quantitative analysis of the assembly of macromolecular complexes during pre-initiation and initiation steps of transcription. Transcription coactivator Mediator (Med) assists in the assembly of a transcription pre-initiation complex. Endogenous Med tagged with Dendra2 forms similarly to RPB1 small (~100 nm) transient (lifetime ~10 s) and large (>300 nm) stable (lifetime > 100 s) clusters in living mouse embryonic stem cells (mESC). The differentiation of mESCs into epiblast-like cells (EpiLCs) had no apparent effect on the small transient Med clusters but reduced the size and the number of large stable Med clusters . Dual-coIn summary, several reviewed SMLM approaches enabled us to study the RNAPII transcription in unprecedented spatial and temporal details and thereby helped to finely refine our mechanistic understanding of the regulation of gene expression. Integration of the information obtained by the SMLM together with biochemistry helped to build a mechanistic model of the localized hierarchical recruitment of co-activators and TFs during the pre-initiation phase followed by the recruitment and formation of RNAPII clusters during the initiation phase and followed by RNAPII leaving the clusters during elongation phase. This hierarchical and dynamic principle of the transcriptional regulation and the discovery of RNAPII forming transient condensates that dictates the productive transcriptional output has progressively changed our mechanistic understanding of gene expression.Since the publication of one of the seminal reviews on SRM in 2010 , twelve


Here, we summarize  X chromosome inactivation (XCI) is the form of dosage compensation used by female cells to balance X-linked gene expression levels between the sexes in mammals . As a reXIST) gene encodes for the long noncoding RNA XIST, which is expressed from the future Xi, coating it in cis [XIST [XIST RNA to its own locus [The initiation of XCI is dependent on a region of the X chromosome known as the X inactivation center (XIC). Inside the XIC, the X-inactive specific transcript  [Polycomb complexes PRC1 and PRC2 carry out histone modifications that are integral to the process of XCI. PRC2 complex catalyzes H3K27me3 . PRC2 cod (ATRX) . It has d (ATRX) . Bivalend (ATRX) .XIST RNA and lays down H3K27me3 [There are two pathways by which the PRC1 complex can be recruited to the Xi, either dependent on PRC2 or independent of the presence of PRC2 , 30–32. H3K27me3 . More reH3K27me3  and polyXist is induced and the enrichment is lost when Xist expression is inhibited [The recruitment of polycomb complexes occurs in the early stages of Xi establishment. Polycomb complexes' enrichment on the Xi is readily detectable when nhibited , 34.HP1 is another protein thought to be important for the establishment and maintenance of the Xi heterochromatin. In humans, there are three isoforms of HP1: HP1-alpha, HP1-beta, and HP1-gamma, all of which contain 3 domains: a chromodomain that can recognize and be recruited to the H3K9me3 on the Xi, a hinge domain, and a chromo shadow domain that is important for dimerization and enrichment of HP1 proteins –37. It hXist mediates the chromosome-wide inactivation and gene silencing is still largely unknown, several factors have been identified to interact with Xist RNA and are essential for Xist-mediated gene silencing. These factors include hnRNP U/SAF-A [Although the exact mechanism of how  U/SAF-A , SHARP/S U/SAF-A –42, and  U/SAF-A .Xist on the Xi [Xist [Xist RNA from the X chromosome, and hnRNP U/SAF-A is essential for establishing the Xi during ES cell differentiation [Xist-mediated gene silencing [The nuclear matrix binding protein hnRNP U/SAF-A is essential for the anchoring of n the Xi . hnRNP  mutagenesis screen to identify genes involved in epigenetic regulation and gene silencing . SMCHD1 Xist or H3K27me3 modification on the Xi, which suggests that SMCHD1 is not involved in the initiation phase of XCI, but the maintenance phase. This is also implied by the fact that, in interphase nuclei, SMCHD1 is enriched on the Xi [XIST is required for the correct localization of SMCHD1 and LRIF1/HBiX1 to the Xi [SMCHD1 is crucial for both random XCI in the embryo and imprinted XCI in the placenta . The losn the Xi . In the n the Xi . It is sn the Xi . Notablyo the Xi , 50.HBiX1/LRIF1 was first identified as a novel nuclear matrix transcription repressor known as ligand-dependent nuclear receptor-interacting factor 1 . It is aSET domain bifurcated 1 (SETDB1), an H3-K9 histone methyltransferase with the highest activity for lysine 9 trimethylation , 54, hasXist to the future Xi; Xist then spreads and coats the whole chromosome. Xist recruits polycomb complexes PRC2 and PRC1 and lays down repressive histone modifications. SHARP/SPEN expels RNA polymerase and inhibits transcription. MacroH2A is then recruited and CpG islands are methylated, and at this point, the establishment stage of XCI is concluded. Heterochromatin proteins such as HP1, SMCHD1, LRIF1, and SETDB1 exert their function in the maintenance stage of XCI and work together to maintain the heterochromatin structure and gene silencing on the Xi.To summarize the process of XCI in regard to the proteins involved and their order of recruitment, hnRNP U/SAF-A is essential for the localization of XIST lncRNA and TSIX lncRNA, which is the antisense transcript of XIST that can mediate the repression of XIST [The XIC is the minimal region on the X chromosome that is both necessary and sufficient to initiate XCI , 60. The of XIST .XIST was first discovered to be important for the XCI in mice and humans in 1991 [XIST lncRNA is 17 kb in length; within XIST are several tandem repeat regions that are conserved. The most highly conserved A repeats were found to be essential for gene silencing during XCI [XIST expression is the first step of inactivation along the future Xi. The localization of XIST to the future Xi is dependent on the transcription factor YY1 and starts with a nucleation center within exon 1 of the XIST locus [XIST follows a stepwise mechanism. It targets gene-rich regions before spreading to intervening gene-poor regions [XIST then recruits polycomb proteins PRC1 and PRC2 to set up repressive histone marks for the maintenance of the inactive chromatin structure [ in 1991 –64. XISTring XCI . The uprST locus . It has  regions . XIST thtructure .XIST is unique to the Xi, the lncRNA TSIX dictates which X chromosome will be the Xa. TSIX is the antisense transcript of XIST and has an antagonistic role in XIST expression. TSIX controls XIST expression by modifying the chromatin state and DNA methylation of the XIST promoter [TSIX on the future Xa [While the expression of promoter , 68. Befuture Xa –72.XIST expression: RepA [A few other lncRNAs have been discovered to be important activators for on: RepA , Jpx [73on: RepA , and Ftxon: RepA .At interphase in human RPE1 cells, the Xi forms a bipartite structure, with all H3K9me3 bands aggregating together towards the nuclear periphery and the H3K27me3 bands more towards the interior of the nucleus . How andWith the exception of the pseudoautosomal regions located at the tips of Xp and Xq, most genes on the X chromosome have been lost on the Y chromosome. As such, males are hemizygous for most X-linked genes. As a consequence, inheritance of an X-linked recessive mutant allele in males will act as dominant and disease onset will be unavoidable. Females are afforded some protection from X-linked recessive disorders due to XCI.Because XCI occurs at a multicell stage, and the choice of which X to silence is random and that decision is made independently in each cell, females are a mosaic, where some cells express the mutant allele, and others express the wild-type allele. If the mutant allele impacts cell growth or survival, these cells will be outgrown giving the appearance of skewing toward the wild-type active X. Skewing of XCI occurs naturally, which can affect disease severity. Fabry disease is one example where there is a mutation in the X-linked lysosomal alpha-galactosidase. The peak of the normal distribution represents equal numbers of cells with either the paternal or maternal X chromosome chosen as the Xi. At either tail end of the normal distribution is the phenomenon called skewed XCI, where at one tail end, it might be asymptomatic, but for the other end of the distribution, there is a severe representation of disease. The direction and degree of skewed XCI influence the phenotype and progression of Fabry disease in female patients .XCI serves as a mechanism for balancing the difference in expression between different sexes in mammalian cells, but there is a certain level of escape from gene repression in both normal and disease cells. A recent systematic survey integrating transcriptomes from 449 individuals across 29 tissues has shown that, besides 683 X-linked genes that are consistently inactivated, there is heterogeneity in expression patterns among different individuals and different tissues . X-linkeX chromosome inactivation is the classic model system to study epigenetic questions. What we learn under the X chromosome inactivation context could also be useful to unravel unsolved problems in autosomes and developmental contexts as well. Research and more mechanistic insight for X chromosome inactivation could assist in developing strategies and therapy for X-linked diseases.


The development of multicellular organisms and the uniqueness of each cell are achieved by distinct transcriptional programs. Multiple processes that regulate gene expression converge at the core promoter region, an 80 bp region that directs accurate transcription initiation by RNA polymerase II (Pol II). In recent years, it has become apparent that the core promoter region is not a passive DNA component, but rather an active regulatory module of transcriptional programs. Distinct core promoter compositions were demonstrated to result in different transcriptional outputs. In this mini-review, we focus on the role of the core promoter, particularly its downstream region, as the regulatory hub for developmental genes. The downstream core promoter element (DPE) was implicated in the control of evolutionarily conserved developmental gene regulatory networks (GRNs) governing body plan in both the anterior-posterior and dorsal-ventral axes. Notably, the composition of the basal transcription machinery is not universal, but rather promoter-dependent, highlighting the importance of specialized transcription complexes and their core promoter target sequences as key hubs that drive embryonic development, differentiation and morphogenesis across metazoan species. The extent of transcriptional activation by a specific enhancer is dependent on its compatibility with the relevant core promoter. The core promoter content also regulates transcription burst size. Overall, while for many years it was thought that the specificity of gene expression is primarily determined by enhancers, it is now clear that the core promoter region comprises an important regulatory module in the intricate networks of developmental gene expression. Drosophila was instrumental in deciphering the identity and function of core promoter composition. The transcription machinery, as well as its regulatory principles, are largely conserved in evolution. Thus, although most of the data discussed here is largely based on findings in Drosophila, many of the underlying principles are applicable to a wider array of species.The uniqueness of each cell type and its various developmental roles in multicellular organisms are largely achieved by distinct transcriptional programs. Transcription initiation is composed of a series of highly conserved, coordinated and complex steps  and the protein  levels. Recent reviews focused on the structure of polymerases, the components of the general/basal transcription machinery and the preinitiation complex . In thisDrosophila and human focused promoters are illustrated in Although the core promoter was previously considered a universal component that works similarly for all protein-coding genes, it is now established that core promoters are divergent in their architecture and function, and each core promoter is rather unique . The coru and BREd, respectively) are bound by TFIIB , which is comprised of Pol II, the basal transcription factors  and promoter DNA. Multiple core promoter elements were identified as the regions bound by distinct PIC components. The upstream and downstream TFIIB recognition elements  and bridge downstream core promoter elements (see below) serve as docking sites for TFIID, which initiates PIC formation by binding to the core promoter. TFIID is composed of TATA box-binding protein (TBP) and 13–14 TBP-associated factors (TAFs)  element, which encompasses the TSS , is the romoters . It is bromoters . Functioromoters . The TCTgulation . This exgulation . While mgulation . Specifiromoters . Motif 1romoters , providiromoters . Notably+1 of the Inr  were discovered ge motif . The trage motif , suggestge motif . Taken tDrosophila species spanning 25–50 million years of evolution, indicated that distinct core promoter elements are associated with different developmental times. Maternally expressed oocyte genes are enriched for DRE and Ohler-1/5/6/7 motifs, whereas zygotic genes expressed through embryogenesis are enriched for Inr, MTE and DPE motifs; larval-related transcripts are enriched for TATA box elements  is a pair-rule gene orchestrating the segmentation phase of Drosophila embryonic development, expressed along the anterior-posterior axis  , specifinogaster . The homE motifs . Importar embryo . The Dro the DPE . Further the DPE . fushi t species . Using Celopment . While telopment , this ise system . Thus, wDrosophila heat shock protein 70 (hsp70) promoter  is essential for embryonic development of . laevis . While T. laevis . Interes. laevis .Xenopus genes involved in mesoderm and organizer specification are TBP family-insensitive  proteins , which are key regulators of embryonic development and differentiation, activate transcription with a preference for a DPE relative to a TATA box (Drosophila CREB-binding protein (dCBP) co-activator and Caudal that enables core promoter-preferential activation. Notably, dCBP co-occupies genomic loci in Drosophila embryos that are bound by Dorsal (TATA box . It is ty Dorsal , in liney Dorsal  and withy Dorsal . Tetheriy Dorsal .Drosophila embryos as well (There is a large body of evidence supporting the similarity between promoters and enhancers, as many enhancers are transcribed and basal transcription machinery components are recruited to enhancers . Transcr as well . The sim as well . A systeDrosophila cells with Cu (Drosophila embryos revealed that enhancers control burst frequency (Drosophila embryo (Drosophila embryos using MS2-based reporters (High-precision methodologies enabled the examination of transcriptional dynamics, which demonstrated the existence of transcriptional bursts . Burst s with Cu . The comrequency . Interesa embryo . Transcreporters . The TATeporters , whereaseporters . The effDevelopmental core Promoter Element. Notably, the downstream core promoter region is transcribed, thus one could speculate that it may exert a regulatory effect at the RNA level. The downstream core promoter region is an important regulatory tier of gene expression, likely conserved and diversified among metazoans. Thus, identification and characterization of downstream motifs in additional mammalian species would likely uncover the regulatory roles of mammalian downstream core promoters and their protein binding factors. Differential core promoter composition may provide a nuanced regulatory switch that recruits the relevant basal transcription factors at the right time during embryonic development to more precisely control the timing of developmental transcriptional programs. Being able to directly visualize and measure transcriptional dynamics will uncover novel insights regarding the functions, mechanisms and definitions of promoters and enhancers, which regulate the complex process known as “transcription.”We envision the core promoter composition as an additional regulatory dimension of the complex developmental GRNs. The DPE acronym, originally defined based on its position, may be indicative of its function as a TJ-G and RF conceptualized the mini-review. AS and HS-S wrote the first draft of the manuscript. AS prepared the figures. AS and TJ-G finalized the review with input from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SNAI1, a zinc finger transcription factor, not only acts as the master regulator of epithelial-mesenchymal transition (EMT) but also functions as a driver of cancer progression, including cell invasion, survival, immune regulation, stem cell properties, and metabolic regulation. The regulation of SNAI1 occurs at the transcriptional, translational, and predominant post-translational levels including phosphorylation, acetylation, and ubiquitination. Here, we discuss the regulation and role of SNAI1 in cancer metastasis, with a particular emphasis on epigenetic regulation and post-translational modifications. Understanding how signaling networks integrate with SNAI1 in cancer progression will shed new light on the mechanism of tumor metastasis and help develop novel therapeutic strategies against cancer metastasis. Tumor metastasis, the spreading of cancer cells from original tumor sites to distant organs followed by development of secondary tumors, is the foremost cause of cancer-related deaths . InitiatCDH1. SNAI1 directly binds to E-boxes present in the CDH1 promoter to transcriptionally repress its expression. On the other hand, SNAI1 also acts as a transcriptional activator. SNAI1 not only enhances mesenchymal markers including fibronectin, collagens, and the matrix degradation enzyme matrix metalloproteinases 2 and 9 (MMP2 and MMP9), it also increases other EMT transcription factors such as TWIST and ZEB1 [Drosophila development through direct binding to the promoters [INK4b, lymphoid enhancer-binding factor (LEF), and cyclooxygenase 2 (COX2) by directly binding on a consensus motif in HepG2 cells stimulated by the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) [SNAI1 was the first discovered and most intensively studied transcription repressor of E-cadherin, a hallmark of EMT encoded by the epithelial gene and ZEB1 ,6. In adromoters . In collte (TPA) ,8,9,10. te (TPA) ,12,13,14te (TPA) . In addiBecause of the reversible plasticity of EMT, epigenetic alternations are required in the EMT process. In eukaryotic cells, genomic DNA interacts with histone proteins and RNA to form chromatin, which holds epigenetic information independent of the DNA genetic data . AlteratHere, we summarize the regulation of SNAI1 with an emphasis on PTMs. Moreover, we describe recent insights into the epigenetic mechanisms of SNAI1-induced cancer metastasis, focusing on the cooperation of SNAI1 with epigenetic regulators.Expression of SNAI1 is governed at multiple levels from gene transcription, post-transcriptional regulation, and translation to PTMs such as phosphorylation, ubiquitination, acetylation, and sumoylation.Drosophila SNAIL lacks the SNAG domain but has a consensus PxDLSx motif and exerts their repressive function through the interaction with the co-repressor c-terminal binding protein (CtBP) [SNAI1 belongs to the SNAIL family which consists of SNAI1 (Snail), SNAI2 (Slug), and SNAI3 (Smuc) . The amin (CtBP) . In the SNAI1 promoter to regulate its expression. Extensive evaluation of this regulation has been covered in other excellent reviews [A diverse repertoire of molecular mechanisms regulating SNAI1 at the transcriptional level have been documented in a variety of organisms. Cytokines, chemokines, and growth factors, such as tumor necrosis factor (TNFα), transforming growth factor (TGFβ), interleukin-6 (IL-6), fibroblast growth factors (FGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) , trigger reviews ,22. Inte reviews . Depleti reviews . In addi reviews .Post-transcriptional control provides a fundamental regulatory mechanism for gene expression. Besides regulation by microRNAs , SNAI1 tBecause of their critical roles in cancer metastasis, much attention has focused on the PTMs of SNAI1. PTMs function in the regulating protein stability, transcriptional activity, and intracellular localization of SNAI1. Among the number of modifications, phosphorylation and ubiquitination represent the best characterized and control a variety of biological activities, such as apoptosis, transcription, metabolism, and stem cell properties. Therefore, gaining deeper insight into the PTMs may help elucidate important steps in cancer metastasis.SNAI1 stability is extensively regulated by phosphorylation . On one SNAI1′s ubiquitination and degradation are controlled by a number of F-box ligases, including β-TRCP1/FBXW1, FBXL14, FBXL5, FBXO11, and FBXO45 . RecentlDeubiquitinases (DUBs) counteract the SNAI1 degradation process to maintain a high level of SNAI1 protein in cancer cells. We recently identified DUB3 as a SNAI1 deubiquitinase that interacts with and stabilizes SNAI1 . IndepenN-acetylglucosamine (O-GlcNAc) modification is a monosaccharide addition. SNAI1 is subject to O-GlcNAc at Ser112 under hyperglycemic conditions [Beyond the well-characterized PTMs of phosphorylation and ubiquitination, at least three other PTMs are involved in regulating SNAI1 protein abundance and activity. First, the sumoylation pathway is very similar to its biochemical analog, ubiquitylation, and regulates diverse cellular processes including transcription and protein stability, chromosome organization, DNA repair, and other cellular processes. TGFβ induces sumoylation of SNAI1 at its lysine (K) 234 residue, which is critical for the EMT-activating function of SNAI1 . Second,nditions . This monditions  , a subunit of SWI/SNF chromatin remodeling complex. This complex increases the DNA methylation and suppresses SNAI1 transcription, leading to a repression of EMT. During hepatocellular carcinoma progression, loss/mutation of ARID2 impairs recruitment of DNMT1 to the SNAI1 promoter. As a result of decreased methylation at the SNAI1 promoter, there is an upregulation of SNAI1 expression that ultimately promotes EMT [DNA methylation involves a covalent attachment of a methyl group to cytosine residues at CpG-rich dinucleotide sequences through DNA methyltransferases (DNMTs). Upon induction of EMT, hypermethylation of the c cancer . Previouc cancer . DNMT1 cc cancer . DNMT1 iotes EMT . These rotes EMT .VEGF and Sox2 promoters to stimulate their expression, leading to endothelium generation and tumor growth [A variety of transcriptional co-activating complexes, which contain lysine acetyltransferase, catalyze lysine acetylation of histone tails. Because acetylation masks the positive charge on lysine residues and weakens the DNA–histone association and relaxes the chromatin structure, histone acetylation is often associated with gene activation. SNAI1 recruits the p300 activator complex to the r growth .SNAI1 promoter, repressing SNAI1 transcription and inhibiting EMT [+-dependent histone deacetylase. Sirt6 interacts with p65 and attenuates NF-kB regulated SNAI1 expression by removing acetyl residues of histone H3K9 and H3K56 in the promoter regions of SNAI1 [Histone deacetylation by histone deacetylase (HDAC) is believed to restrict gene transcription because it reveals the positive charge of lysine and permits the DNA–histone interaction. HDACs, in particular HDAC1 and HDAC2, are often recruited by EMT-TFs to gene promoter regions and form protein complexes to deacetylate histones and silence expression of epithelial gene factors. For instance, SNAI1 mediates recruitment of the HDAC1/2 that contain Sin3A or NuRD repressor complexes to inhibit CDH1 expression by deacetylation of histones H3 and H4. This effect was abolished by treatment with the HDAC inhibitor Trichostatin A (TSA) ,72. Inteting EMT . Recentlting EMT . HDAC8 iof SNAI1 .N-aminoacetyl groups of nucleosomal histone lysines and recruit histone modifiers and transcriptional/remodeling factors to gene promoters; these processes promote upregulation or repression of gene expression. Ever increasing studies in different cancer cells have demonstrated the contribution of BRDs to cancer progression [The bromodomain-containing proteins (BRDs) are acetylation readers that bind to ε-gression . For insgression . In addigression . TherefoCDH1 promoter for transcription silencing. Therefore, inhibition of G9a reduces promoter H3K9me2 as well as DNA methylation which abrogates EMT and tumor metastasis [CDH1 promoter to repress its transcription. EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), promotes transcriptional silencing of CDH1 by H3K27me3 [SNAI1 that enhances SNAI1 de-repression, consequently promoting EMT [Histone lysine methylation is catalyzed by lysine methyltransferases, which directly recruit or inhibit the recruitment of histone-binding proteins. Usually, H3K9 and H3K27 methylation is associated with transcriptional repression, while H3K79 is often linked with gene activation. G9a is responsible for the transcriptionally repressive modification of H3K9. In aggressive lung cancer cells, G9a is preferentially expressed, and its elevated expression correlates with poor prognosis. G9a represses a cell adhesion molecule EPCAM, which stimulates EMT and cancer metastasis by catalyzing H3K9me2 on its promoter . In breatastasis . MeanwhiH3K27me3 ,83. EZH2H3K27me3 . DOT1L cH3K27me3 . In breating EMT .SNAI1 promoter to activate the transcription of SNAI1 during TGFβ-induced EMT [Histone lysine-specific demethylase 1 (LSD1) functions as an epigenetic regulator by removing methyl groups on the transcription-activating H3K4 or repressing H3K9 residues through an amine oxidase reaction ,88. LSD1uced EMT . In addiuced EMT .Given the important role of SNAI1 in driving cancer progression, targeting SNAI1 would be an attractive anticancer therapeutic approach. However, the development of small molecules to inhibit SNAI1′s functions is hindered as there is no clear “ligand-binding domain” for targeting SNAI1. However, other strategies have been successfully attempted. First, the E-box, a SNAI1-binding site, was chosen as a target. A Co(III) complex conjugated to a CAGGTG hexanucleotide was synthesized. This complex binds to SNAI1 and prevents any interaction with DNA, thus reducing the invasive potential of tumor cells . Second,Proteolysis-targeting chimeras (PROTACs) that hijack the ubiquitin-proteasome system for targeted protein degradation have expanded significantly in years . This teSNAI1 as the key EMT regulator plays important roles in invasion and metastasis. The molecular events mediated by SNAI1 are of interest as therapeutic targets, in particular for resistant metastatic tumors. Although direct targeting of SNAI1 is unsuccessful, identifying inhibitors for PTMs of SNAI1 hold significant potential, and thus are a high priority in the development of future cancer treatments. Indeed, many pharmacological approaches, including chemical inhibitors and monoclonal antibodies that target these modification enzymes including deubiquitinase and kinase, have been devised and show promise for the treatment of tumor metastasis . FurtherIn addition, because of the intimate connection between SNAI1 and chromatin-remodeling enzymes, targeting the epigenetic enzymes to reverse the EMT process is also an efficient and promising approach . Indeed,