Across all species, transcription factors (TFs) are the most frequent targets of SUMOylation. The effect of SUMO conjugation on the functions of transcription factors has been extensively studied in animal systems, with over 200 transcription factors being documented to be modulated by SUMOylation. This has resulted in the establishment of a number of paradigms that seek to explain the mechanisms by which SUMO regulates transcription factor functions. For instance, SUMO has been shown to modulate TF DNA binding activity; regulate both localization as well as the abundance of TFs and also influence the association of TFs with chromatin. With transcription factors being implicated as master regulators of the cellular signalling pathways that maintain phenotypic plasticity in all organisms, in this review, we will discuss how SUMO mediated regulation of transcription factor activity facilitates molecular pathways to mount an appropriate and coherent biological response to environmental cues. Indeed, numerous substrates for SUMO modifications have been linked to proteins involved in the regulation of transcription/chromatin dynamics [Transduction of environmental cues into cellular signalling is critical for the survivability and development of adaptive responses in any organism. An individual organism can be simultaneously subjected to multiple environmental and physiological cues and a dynamic network of interconnected signal transduction cascades decode and integrate these signals into a coherent response. Due to their sessile nature, plants heavily depend on an intricate network of signalling pathways that allows them to perceive the changes in their environment and mount an appropriate adaptive response to maintain phenotypic plasticity , 2. Trandynamics and transdynamics . Recent dynamics . SUMO codynamics .Irrespective of whether SUMO conjugation directly or indirectly impacts the function of a TF in the context of its ability to bind DNA, the process of SUMO conjugation to transcription factors profoundly impacts the expression of downstream target genes and alters the fate of a signalling pathway. This review aims to discuss the implications of SUMO conjugation as a mechanism to modify and regulate the transcriptional output of cellular signalling pathways in response to environmental cues, with emphasis on the SUMOylation of transcription factors involved in the various cellular signalling pathways. We will first set the scene by having a brief overview of SUMO and its isoforms and the key players of the SUMO conjugation/ deconjugation machinery in plants. We will also assess how the pool of SUMO proteins and the turnover of SUMOylated proteins are maintained in a cyclical manner (SUMO cycle) in plants. We will then finally examine the well- documented examples of SUMOylation mediated modulation of the functions of transcription factors and its outcome on various cellular signalling processes in plants and also assess whether these examples in plants re-enforce the established paradigms of SUMOylation mediated modulation of gene expression and cell signalling.Saccharomyces cerevisiae, mammals and plants. The indispensability of SUMO proteins for eukaryotic cell viability and survival is evident from the fact that deletion of the only SUMO isoform, SMT3, in yeast cells results in the loss of cell viability[Arabidopsis thaliana sumo1sumo2 deletion mutants are embryonic lethal[SUMO proteins are ubiquitously found in all eukaryotes including single-celled eukaryotes viability, 13 and ic lethal, 15. SUMic lethal, 17. Theic lethal. Howeveric lethal. Evidentic lethal–24. Thus. This diversification between the SUMO isoforms may have implications on the dynamics of SUMOylation in plants as it involves residues that perform key molecular functions involving interactions with the key enzymes of the SUMO conjugation system, E1 and E2 and as well as interactions with the SUMO-interacting motif (SIM). SIMs are characterized by hydrophobic residues flanked by acidic residues or residues that can be phosphorylated [sumo3 knock out plants are not impaired in resistance to infection by Pseudomonas syringae pv tomato DC3000 (PstDC3000) [The existence of multiple distinct isoforms of SUMO confers diversity to the process of SUMOylation of target proteins and also contributes to the dynamicity of molecular consequences of SUMOylation. Modulation of the function of a target protein by a specific isoform of SUMO may act as a critical regulatory point in a molecular signalling pathway where identification of the SUMO isoform conjugated to the target protein may act as a “specificity checkpoint” that allows or disallows the downstream steps of the molecular pathway to proceed. Eight SUMO isoforms are encoded by the Arabidopsis genome and amongst them, only the SUMO1, 2, 3 and 5 isoforms are expressed –27. SUMOorylated , 30. SUMorylated  and are orylated . HowevertDC3000)  suggestitDC3000) , implyin. On the other hand, although T-DNA mutants of the SAE1a isoform are viable [The E1 SUMO activating enzyme is a heterodimer consisting of a large subunit SAE2 (SUMO activating Enzyme2) and a small subunit SAE1 (SUMO activating Enzyme1). There are two isoforms of SAE1, SAE1a or SAE1b and either of the two isoforms can be involved in the formation of the heterodimer with SAE2. SAE2 in turn consists of four functional domains: adenylation, catalytic cysteine, UFD (ubiquitin-fold domain) and C-terminal domains. The adee viable , kinetice viable . Moreovee viable . These fSCE1 and SAE2 (sce1 and sae2) are embryo lethal in Arabidopsis with the embryos being arrested in early embryonic stages  and this indicates the indispensability of both E1 and E2 for SUMO modifications of target proteins[The SUMO E2 conjugating enzyme is involved in the transfer of activated SUMO to a consensus acceptor lysine residue of the target substrate protein. Like ubiquitin-conjugating enzyme, SUMO E2 conjugating enzymes also display a characteristic fold consisting of four α-helices and four β-strands containing a signature histidine-proline-asparagine (HPN) tripeptide motif followed by the catalytic cysteine residue at the 7th or 8th position and a tryptophan residue at the 16th position (up to 29th position) from the C-terminal end of the HPN motif. Like in proteins. SCE1 sh proteins. The mRN proteins. Apart f proteins. Moreove proteins, 37, 38.siz1 mutation phenotype characterized by growth retardation, ABA hypersensitivity and cold sensitivity. Moreover, the PHD domain of SIZ1 also interacts with the H3K4 methyltransferase, ATX1 and this interaction is proposed to suppress ATX1 methyltransferase functions. While still being subject to further validation, but these recent studies indicate that the PHD domain of SIZ1 may function as an essential sensor of the epigenetic landscape of chromatin and allow SIZ1 to modulate transcriptional pathways dependent on the status of chromatin. In contrast to SIZ1, MMS21 /HPY2 (High ploidy2) ligases only possess the SP-RING domain and a putative SIM. HPY2 is implicated in several biological processes in Arabidopsis including stem cell maintenance [siz1mms21 mutations are embryo lethal [SUMO E3 ligases facilitate the transfer of activated SUMO from E2 to the target substrate although the requirement of E3 is not essential for in vitro SUMO conjugation to some substrates. SUMO E3 ligases do not form a thioester bond with SUMO but bind to the SUMO conjugating enzyme E2 and functions as a bridge or adaptor between E2 and the substrate to promote the transfer of SUMO to the substrate . In animntenance , cell cyntenance , 57, gamntenance , flowerintenance  and drountenance . Double o lethal  and geneThe fate of the target protein that is SUMOylated is also determined by the multiplicity of the SUMO molecules attached to the target protein, which can also be polymeric in nature . As discIn plants, four major classes of cysteine proteases are described in the MEROPS database: cysteine-, serine-, aspartate-, and metallo-proteases. The protease superfamily comprises 2% of the coding genes in plants . Cystein. The Arabidopsis genome has been predicted to encode eight ULPs and six amongst them have been characterized as SUMO proteases. ESD4 (early in short days 4) was the first characterized ULP in Arabidopsis, with the protein being identified first by Reeves et al. [-1 mutants also have an increased abundance of SUMO conjugates and reduced levels of free SUMO. The yeast genome encodes for two ULP proteins, ULP1 and ULP2, and the yeast ULP1 (ubiquitin-like protease 1) protein was the first isolated SUMO protease through an activity-based screen of S.cerevisiae [Arabidopsis thaliana SUMO proteases with sequence similarity to yeast ULP1. Twelve genes were found from this search and these genes were further classified into three sub-families, with two subfamilies more related to yeast ULP1 and the third more similar to yeast ULP2. ELS1 (ESD4-like SUMO protease1) and ELS2 were identified from this study and were name ULP1a and ULP1b, respectively. The OTS1 and OTS2 ULPs were also initially identified from the study by Kurepa et al. [Arabidopsis SUMO protease1). The endopeptidase activity of SPF1 was confirmed through assessment of the cleavage of SUMO from SUMOylated FLC by wild type SPF1 [Ubiquitin like-specific proteases (ULPs) are members of the CE clan of cysteine proteases and belong to the ULP family. ULPs mediate SUMO maturation and deconjugation of SUMO from the target proteins through their endopeptidase and isopeptidase activities, respectively  and posss et al.  through s et al.  by sequerevisiae . BLAST-brevisiae  resulteda et al. , and wera et al.  and Chosa et al. . The stua et al.  also dema et al. . These pa et al.  and Konga et al.  and thesa et al. ; while Ka et al.  renamed ype SPF1  and thisype SPF1 . FUG1 waype SPF1 , 78; ULPype SPF1 , althougype SPF1 . Group A.The DeSI SUMO proteases, DeSI1 (deSUMOylating isopeptidase 1) and DeSI2 (deSUMOylating isopeptidase2) constitutes a novel type of SUMO proteases lacking sequence similarity to ULP enzymes and were identified first as a SUMO protease in mouse . The micUSLP1 is a recently identified SUMO protease in humans that has been shown to bind to SUMO2 but not to ubiquitin and has also been shown to possess peptidase activity and some chain editing activity . CurrentψKXE/D; ψ denotes a large hydrophobic residue, K denotes the target lysine residue, X denotes any amino acid residue and E/D denotes glutamate or aspartate. While this step of SUMO conjugation to target substrate proteins can occur unaided, it is often mediated by SUMO E3 ligases that can interact with SCE1 and facilitate the transfer of SUMO from the SCE1-SUMO intermediate to the target protein. While the concerted action of the biochemical steps discussed above results in SUMOylation of the target proteins, the cyclical nature of the SUMO cycle is conferred upon it by the action of SUMO proteases. SUMO proteases can cleave SUMO from target substrate proteins; thereby not only making SUMOylation a reversible modification but also acting as a critical determinant for the maintenance of the pool of free SUMO. Secondly, with the cellular pools of unconjugated SUMO being very low [The dynamicity of SUMOylation as a post-translational modification arises at least in part from the fact that the process is cyclical. Essentially, a cascade of enzymatic steps that are biochemically similar to ubiquitination is involved in the SUMO cycle by which both the turnover of conjugated and non-conjugated free SUMO and the pool of SUMO conjugated protein is maintained during the course of the cell cycle and in response to exogenous stimuli Fig. . These mvery low , they prvery low , 92, 93 very low  and thusIn the following sections, we will assess how SUMOylation might influence the functional outcome of a signalling pathway in response to environmental cues through SUMOylation dependent modulation of transcription factor activity in plants by a detailed examination of the well-documented examples. As discussed earlier, SUMO conjugation can influence TF functions through multiple ways, either through “direct mechanisms” involving modulation of the DNA binding properties of the TF Fig.  or throuArabidopsis thaliana genome, as much as 1500 are considered to be involved in stress-responsive gene expression [CBF/DREB1 expression) in response to cold stress provides a typical example of SUMOylation driven alteration of a specific transcriptional output in response to environmental cues facilitated by the protection of the key transcription factor involved in the process from degradation by ubiquitination and by alteration of the target specificity of the transcription factor [Arabidopsis plants devoid of the SUMO E3 ligase, SIZ1 exhibit reduced expression of DREB1/CBF, especially DREB1A/CBF3. However, the expression of ICE1 is unaltered in siz1 mutant plants indicating that SIZ1 functions downstream of ICE1 expression [CBF3 and ice1 mutation abolishes the expression of CBF3 as well as genes downstream of CBF3 [DREB1/CBF3 and consequently the CBF regulon thereby leading to cold acclimatization and increased freezing tolerance. Moreover, SUMO conjugation to ICE1 also impedes the RING type E3 ubiquitin ligase, HOS1  mediated polyubiquitination of ICE1[CBF3/DREB1 expression, it also represses the expression of MYB15 [myb15 expression during cold exposure in both ice1 [siz1 mutants [myb15 and it will be interesting to further assess whether SUMOylated ICE1 attenuates myb15 expression by directly binding to the myb15 promoter as a transcriptional repressor or indirectly attenuates myb15 expression by activating other downstream genes that can repress myb15 expression or through interactions with other proteins that may presumably be chromatin modifiers that can alter the myb15 chromatin landscape into a more repressive state for transcription. Similarly, since the ICE1 (K393R) substitution does not impact the transactivation activity of ICE1 in Gal4 assays but leads to the down-regulation of CBF3/DREB1 in ICE1 (K393R) overexpressing plants, the possibility that the SUMOylation of ICE1 may influence the capacity of ICE1 to transform the chromatin landscape of CBF3/DREB1 to a state that facilitates transcription cannot be negated. Nevertheless, repression of myb15 and induction of DREB1/CBF3 by SUMO conjugated ICE1 allows SUMOylation of the same transcription factor to result in both the induction as well as the repression of different sets of target genes of the transcription factor [. As a future perspective, identification of the specific SUMO proteases that may be implicated in the deSUMOylation of ICE1 to regulate the functions of ICE1 in the absence of cold stress will further enhance our understanding of how the regulation of the expression of cold responsive genes is intricately dependent on the dynamics of post- translational modification of transcription factors that can activate or repress cold responsive genes. Another abiotic stress related MYB transcription factor, whose activity is regulated by SUMOylation is PHR1 (phosphate starvation response1). PHR1 is a key molecular determinant required for phosphate starvation dependent responses and controls a subset of genes whose expression is activated by phosphate (Pi) limitation by binding to DNA cis elements known as P1BS (PHR1 specific binding sequence) [Arabidopsis PHR1 and the nuclear localization status of the PHR1 protein in response to any alterations in Pi status provided the initial clue that PHR1 activity might be regulated by post translational modifications [Arabidopsis. Lysine residues at position 261 and 372 of AtPHR1 are crucial targets for SIZ1 mediated SUMOylation of AtPHR1 and K261R and K372R substitutions impede PHR1 SUMOylation. SUMOylation by SIZ1 has been suggested to stabilize the abundance and activity of PHR1 and accelerate its binding affinity to the P1BS motif present at the regulatory region of its downstream targets such as AtIPS1 (INDUCED BY PHOSPHATE STARVATION 1) and AtRNS1 (RIBONUCLEASE 1) [OTS1/OTS2 (ULP1C/ULP1D) SUMO protease genes in negatively influencing drought stress resistance has been previously demonstrated in Arabidopsis, where ulp1culp1d double mutants exhibit enhanced drought resistance in comparison to wild type plants [OsOTS1 results in increased drought sensitivity while OsOTS1 depleted transgenic lines exhibit drought tolerance in pot grown plants [Arabidopsis [AOX1a  and up-regulate its expression. Moreover, MYB30 K283R mutant fails to rescue the salt sensitive phenotype of myb30-2 mutant, again indicating the indispensability of SUMO conjugation for MYB30 activation of AOX1a expression, which in turn facilitates the maintenance of cellular redox homeostasis via AOX1a and confers salt tolerance.Plant’s response to abiotic stresses involves the activation of several genes whose products confer stress tolerance and on the other hand repression of several genes whose products can interfere negatively with stress tolerance. The activation and repression of these distinct classes of genes are mediated by the utilization of the activity of dedicated stress-responsive transcription factors, whose target genes form a regulon that is involved in the activation/repression of genes associated with abiotic stress responses. Amongst the described TFs encoded by the pression . As indipression , 97. Thepression , high tepression , 99, frepression , droughtpression , excess pression , oxidatipression , 104, etpression  indicaten factor . The Dehn factor –107, andn factor , 109. Whpression . ICE1 is of CBF3 . Indeed,n of ICE1 and protn of ICE1. Howevern of ICE1. Moreoveof MYB15 . MYB15 iof MYB15 . The suboth ice1  and siz1 mutants  also indn factor –116. As equence) –119.The ications . Indeed,LEASE 1) . In the LEASE 1)  demonstrLEASE 1)  interacte plants  and as wn plants . The stun plants , clearlybidopsis . These tbidopsis ; a membebidopsis , 123 andbidopsis . In respin planta [CPD indicated by chromatin immunoprecipitation assays [The plant-specific steroid hormone, brassinosteroid (BR) is implicated in the regulatory control of a variety of physiological processes ranging from seed development to flowering and senescence –127. Recn planta .SUMOylatn assays .abscisic acid insensitive 5) plays a central role in abscisic acid signalling by regulating the expression of downstream genes that harbour the ABSCISIC ACID RESPONSE ELEMENT (ABRE) motif at their promoter regions [siz1 mutant plants display hypersensitivity to ABA for inhibition of seedling primary root growth and seed germination and hyper induction of ABI5 downstream target genes such as RD29A, RD29B, AtEm6, RAB18, ADH1. Furthermore, expression of a mutant K391R transgene of ABI5 that cannot be SUMOylated in abi5 loss of function mutants also resulted in increased ABA hypersensitivity and ABI5 downstream target gene expression [ABI5 gene in response to ABA. With a relatively large number of plant SUMO proteases displaying target protein specificity, future identification of specific SUMO proteases implicated in the regulation of ABI5 functions will provide the final missing piece to the puzzle of regulation of ABI5 functions by SUMOylation/deSUMOylation. SUMOylation of the transcription factor MYB30 by SIZ1 also has implications on ABA signalling [. Like ABI5, SUMOylation of MYB30 at lysine 283 by SIZ1 prevents its degradation and increases the stability of the protein. However, in contrast to ABI5, SUMOylation of MYB30 positively impacts its transcriptional activity in planta as evidenced from the fact that in contrast to MYB30, expression of MYB30K283R does not rescue the expression of downstream target genes TAT3, LOX3, BGL2, bHLH, COR15b and COR413 and does not fully restore ABA sensitivity in myb30 plants. While previously, the importance of regulation of the transcriptional activity of MYB30 by the degradation of the protein by RING E3 ligase MIEL1 (MYB30-Interacting E3 Ligase1) mediated ubiquitination has been demonstrated in hypersensitive cell death response and defense in plants [The ABA- responsive bZIP transcription factor, ABI5 , aminocyclopropane-1-carboxylic acid synthase (ACS), the auxin-responsive Gretchen Hagen3 (GH3) family and small auxin upregulated RNA (SAUR) [LBD16. Increased occupancy of ARF7-4K/4R-GFP (a GFP tagged non SUMOylatable version of the protein where the target lysine residues for SUMO conjugation are mutated to arginine) at the promoters of its downstream targets LBD16 and LBD29 in comparison to wild type ARF7-GFP indicates that SUMOylation of ARF7 negatively regulates its auxin-induced DNA binding activity [4K/4R-GFP at the promoters of LBD16 and LBD29 in comparison to ARF7-GFP also indicates that SUMOylation positively influences ARF7 promoter clearance. The negative impact of SUMOylation on the transcriptional activity of ARF7 is attributed to the function of SUMO as an essential mediator for the interaction of ARF7 with the Aux/IAA (indole -3-acetic acid) repressor protein, IAA3. The SUMO interacting motif (SIM) of IAA3 is critical for the interaction of IAA3 with SUMOylated ARF7 and this interaction results in the creation of a transcriptional repressor complex that can block the expression of auxin-responsive genes on the airside of roots, where there is non-availability of water. Conversely, maintenance of ARF7 in its non –SUMOylated form on the water contact side putatively by the action of the SUMO protease OTS1, allows induction of ARF7 target genes like LBD16 to trigger lateral root organ initiation. The regulation of ARF7 transcriptional activity by SUMOylation illustrates how SUMO conjugation can influence the function of a TF by modulation of the interactions of the TF with a co- repressor protein resulting in a change in the status of the TF from an activator to a component of a repressor complex. SUMOylation can also have the opposite effect on the status of a TF by promoting the association of the TF with transcription co-activators as evident from the SUMOylation dependent regulation of the function of the master regulator of Salicylic acid (SA) responsive genes, NPR1(NON EXPRESSOR OF PATHOGENESIS RELATED GENES1), which is discussed in detail in the following section.The plant hormone, auxin, regulates lateral root development  apart frA (SAUR) , 162. SUA (SAUR) . “HydropA (SAUR) , 165. ARA (SAUR) , 166–168activity . Indeed,PR genes is essential for SAR and NPR1 plays a critical role in this process as a positive regulator of PR gene expression and thereby SA response. Indeed, Arabidopsis mutants deficient in NPR1 exhibit reduced expression of PR gene and increased susceptibility to pathogens [NPR1 in wild type Arabidopsis thaliana ensures a quick response to salicylic acid. In the absence of SAR, NPR1exists as an oligomer formed through intermolecular disulfide linkages and is sequestered from transport to the nucleus. SA-regulated immunity involves SAR induction triggered monomerization of NPR1 and localization of NPR1 to the nucleus [PR gene expression by interacting with a class of bZIP transcription factors called TGAs (TGACG motif binding protein family) [PR1 by localizing to the WRKY DNA binding motif, W box present at the promoter of PR1. Conjugation of SUMO3 to NPR1 upon SA induction, on the other hand, promotes the dissociation of NPR1 from both WRKY and the W-box motif as evident from ChIP assays where conversion of NPR1 to a non SUMOylatable form results in its constitutive localization to the W box motif of PR1 promoter. Parallelly, SA induced SUMOylation of NPR1 dramatically increases the association of NPR1 with the as-1 element present at the PR1 promoter through SUMO3 mediated interactions of NPR1 with TGA3 [NPR4 mediated ubiquitination of NPR1 and this prevents the activation of immune response genes [NPR3. Although paradoxical, this degradation of NPR1 mediated by the alternate E3 ligase CRL3NPR3 is necessary for the full induction of the target genes of NPR1 [S11D/S15D exhibits enhanced interaction with SUMO3 that leads to further SUMOylation. On the other hand, SUMO3 also promotes phosphorylation of Ser11/15 of NPR1, and thus by these mechanisms SUMOylation amplifies the transcriptional output from the SA signal by coordinately inducing the function of NPR1 as a transcriptional co-activator and by maintaining the turnover of NPR1that is in turn required for the full activation of its downstream targets. SIZ1 mediated SUMOylation of the transcriptional co-repressor TOPLESS-RELATED1 (TPR1) is also implicated in plant immune response [DND1 (DEFENSE NO DEATH1) and DND2. In this regard, SUMOylation of TPR1 at lysine 282 and lysine 721 residues influences TPR1 mediated plant immune response by inhibiting its co-repressor activity putatively by repressing its interaction with HDA19. Since histone deacetylases remove acetyl groups from ε-N-acetyl-lysine residues from histones thereby allowing histones to wrap DNA more tightly, inhibition of the interaction of HDA19 and TPR1 may confer a more “open” chromatin state at the regulatory regions of the target genes of TPR1 thereby facilitating the transcription of these genes such as DND1 and DND2. Thus evidently, SUMOylation mediated modulation of TPR1 functions indicate that in plant systems also SUMOylation can alter the capacity of the TF to interact with chromatin dependent transcriptional regulators like histone deacetylases (HDAC).The low molecular weight phenolic compound, Salicylic acid (SA) is a critical signalling molecule in plant defense response, required for both local and systemic immunity in plants, a phenomenon known as systemic acquired resistance (SAR), and is therefore widely regarded as the key plant immunity hormone. The NPR1 (NON EXPRESSOR OF PATHOGENESIS RELATED GENES1) protein acts as a master regulator of SA signalling as it connects the initial step of the SA signalling pathway i.e. the perception of SA, to the final stages of the pathway involving activation of the expression of pathogenesis-related (PR) genes. The expression of athogens , 170 and nucleus , where i family) –175. Wit family) . Unmodifith TGA3 . In the se genes –178. An  of NPR1 , 178. Evresponse . TPR1 fuga1-3 SCARECROW) family of transcriptional regulators that mediate gibberellin (GA) signalling and function as key repressors of molecular pathways that are governed by GA [ga1-3) has been shown to allow plants to control growth independently of gibberellin [The plant hormone, gibberellin, mediate regulation of many growth and developmental aspects throughout the life cycle of plants that includes promotion of cell division and elongation, elongation of stem and roots, bolting, control of seed dormancy and germination, and promotion of flower and fruit development and responses to biotic and abiotic stresses –183. Theed by GA –187. Reced by GA –192 and ed by GA . For insed by GA . A direced by GA –202. SUMberellin . Amongstberellin  and nullberellin , SUMOylaberellin  and drouberellin  stress. FLC (Flowering locus C) gene, which encodes a MADS box transcription factor [FLC regulates almost all the major genetic pathways implicated in floral transition by repression of the key players of the different pathways that are involved in the activation of the floral meristem identity genes, many signals that control flowering converge in the regulation of FLC expression. One of the key proteins that is involved in the regulation of FLC expression is the autonomous pathway protein, FLOWERING LOCUS D (FLD). FLD is the plant ortholog of human KIAA0601/LYSINE-SPECIFIC HISTONE DEMETHYLASE1 (LSD1) and it represses FLC expression by facilitating deacetylation of histone H4 in FLC chromatin [FLC chromatin in fld-6 protoplasts transiently expressing either HA:FLD or HA: FLDK3R (constitutively non SUMOylatable) indicated that the levels of FLC mRNA and the H4 acetylation levels at FLC chromatin were significantly reduced in protoplasts expressing the non SUMOylatable version of FLD (HA: FLDK3R) as compared to protoplasts transiently expressing HA:FLD [FLC transcript accumulation and H4 hyperacetylation at the first intron of FLC [In higher plants, flowering involves the transition of a vegetative meristem producing leaves and stems to a floral meristem producing flowers, that requires the coordinated and sequential functions of a wide array of transcription factors and is regulated through a complex network of both genetic and epigenetic pathways. The regulation of flowering time in Arabidopsis can be controlled by four major genetic pathways. The long-day and the vernalization pathways mediate floral transition in response to environmental cues, whereas the other two pathways namely the autonomous pathway and the GA pathway function independently of environmental cues. The autonomous pathway promotes flowering under all conditions and GA pathway is required for flowering during non-inductive short-day conditions. Irrespective of the pathway employed, the final output of these genetic pathways converge in the induction of a key set of genes known as floral meristem identity genes, which promotes the specification of floral meristems on the flanks of the shoot apical meristem –212. Regn factor  serves ahromatin , 215. Evg HA:FLD . The resn of FLC . Thus, STable SND1/NST1-mediated transcriptional networks for SCW formation in fiber cells [Increasing pieces of evidence indicate that the indispensability of SUMOylation as a post-translational modification implicated in growth, development, maintenance of phenotypic plasticity and dynamic responses to changes in the environment in plants, is largely due to SUMOylation of TFs that are essential for these processes. The list of biological processes that are influenced by SUMOylation/ deSUMOylation of transcription factors that are implicated in the molecular signalling pathways regulating the concerned biological processes in plants are ever-growing. For instance, apart from the biological signalling pathways discussed above, recent studies have indicated that SUMOylation affects the circadian clock and secondary cell wall formation in Arabidopsis. SUMOylation was shown to suppress the DNA binding activity of the morning phased central plant clock transcription factor CIRCADIAN CLOCK ASSOCIATED (CCA1)  while SIer cells . Large ser cells  and refeer cells  for furt


Cockayne syndrome group B protein (CSB), a member of the SWI/SNF superfamily, resides in an elongating RNA polymerase II (RNAPII) complex and regulates transcription elongation. CSB contains a C-terminal winged helix domain (WHD) that binds to ubiquitin and plays an important role in DNA repair. However, little is known about the role of the CSB-WHD in transcription regulation. Here, we report that CSB is dependent upon its WHD to regulate RNAPII abundance at promoter proximal pause (PPP) sites of several actively transcribed genes, a key step in the regulation of transcription elongation. We show that two ubiquitin binding-defective mutations in the CSB-WHD, which impair CSB’s ability to promote cell survival in response to treatment with cisplatin, have little impact on its ability to stimulate RNAPII occupancy at PPP sites. In addition, we demonstrate that two cancer-associated CSB mutations, which are located on the opposite side of the CSB-WHD away from its ubiquitin-binding pocket, impair CSB’s ability to promote RNAPII occupancy at PPP sites. Taken together, these results suggest that CSB promotes RNAPII association with PPP sites in a manner requiring the CSB-WHD but independent of its ubiquitin-binding activity. These results further imply that CSB-mediated RNAPII occupancy at PPP sites is mechanistically separable from CSB-mediated repair of cisplatin-induced DNA damage. ERCC6 gene encoding CSB [The Cockayne syndrome group B protein (CSB), a member of the SWI/SNF2 (switch/sucrose non-fermenting) family, is a multifunctional protein that participates in a wide range of cellular processes. CSB was first described for its role in transcription-coupled nucleotide excision repair (TC-NER) ,2. Impaiding CSB ,4,5. Howding CSB ,8, oxidading CSB , mitochoding CSB ,11, cellding CSB , telomerding CSB ,14 and Dding CSB ,16,17, iding CSB ,19 as weding CSB ,21. It hding CSB ,23,24.CSB resides in an elongating RNA polymerase II (RNAPII) complex ,26,27. RCSB contains a N-terminal region, a central ATPase domain and a C-terminal region. The last 76 amino acids of CSB constitute a winged helix domain (WHD) ,35. ThisACTB, GAPDH and RPL13A genes. In addition, we demonstrate that two cancer-associated mutations, arginine to glutamine at position 1467 (R1467Q) and glycine to arginine at position (G1484R), both of which are located on the opposite side of the CSB-WHD away from its ubiquitin-binding pocket, impair its ability to promote RNAPII occupancy at PPP sites of ACTB, GAPDH and RPL13A genes, providing the first evidence that CSB somatic mutations affect RNAPII association with PPP sites. Taken together, these results suggest that the CSB-WHD mediates RNAPII association with PPP sites in a manner independent of its ubiquitin-binding activity. These results further imply that CSB-mediated RNAPII association with PPP sites is mechanistically separable from CSB-mediated repair of cisplatin-induced DNA damage. In this report, we show that CSB is enriched at PPP sites and promotes RNAPII association with PPP sites of several actively transcribed genes through its WHD. We show that the previously reported ubiquitin binding-defective mutations in the CSB-WHD, which impair CSB’s ability to promote cell survival in response to treatment with cisplatin, do not affect its ability to mediate RNAPII association with PPP sites of ACTB, GAPDH, RPL13A, CALM3, VCL, TEFM, and MRPL21 are actively transcribed genes that have previously been used for studies of RNAPII occupancy [ACTB, GAPDH and RPL13A genes since these three genes contained a higher level of RNAPII at their PPP sites than the other four genes . ChIP-qPCR analysis showed that overexpression of Myc-CSB in GM16095 cells enhanced RNAPII occupancy at PPP sites of   . ChIP-qPand VCL) B. The deand VCL) A. Formald RPL13A C, indicad RPL13A C, suggesACTB, GAPDH and RPL13A genes in U2OS CSB-KO cells , alanine to serine at position 1445 (A1445S), arginine to leucine at position 1462 (R1462L), arginine to glutamine at position 1467 (R1467Q) and glycine to arginine at position 1484 (G1484R)  A, which (G1484R) B. In add(G1484R) A. We rea(G1484R) C,D. ChIPKO cells A,B. ClonKO cells C–E, suggACTB, GAPDH and RPL13A genes. Firstly, both D1425A and F1437D mutations, which have previously been reported to not only abrogate binding of the CSB-WHD to ubiquitin but also sensitize cells to treatment with UV [ACTB, GAPDH and RPL13A genes. Secondly, a cancer-associated D1425N mutation of CSB, which sensitizes cells to cisplatin, does not affect RNAPII occupancy at PPP sites of ACTB, GAPDH and RPL13A genes. D1425 has been reported to be directly engaged in binding to ubiquitin [ACTB, GAPDH and RPL13A genes. Our finding suggests that the surface of the CSB-WHD where R1467 and G1484 reside may play a role in regulating RNAPII association with PPP sites of ACTB, GAPDH and RPL13A genes, which requires further investigation. It has been suggested that CSB tracks paused RNAPII on non-damage templates to stimulate transcription ,43, howe with UV , have libiquitin . ThirdlyWhile much attention has been given to CS-causing CSB mutations over the past decades, little is known about CSB somatic mutations that have been identified in cancer cell lines and tumor samples. The work presented here has provided the first evidence that CSB somatic mutations affect CSB function in cultured cells. Increasing evidence suggests that CSB plays a role in cancer prognosis and treatment ,39,40,41ACTB, GAPDH and RPL13A genes is mechanistically separable from CSB-mediated repair of cisplatin-induced DNA damage. Our finding adds to the growing list of evidence that CSB function in various nuclear processes is distinctively regulated from one another. Our finding that five of six cancer-associated CSB mutations, D1425N, Q1444H, A1445S, R1462L and G1484R, differentially affect CSB’s ability to regulate RNAPII association with ACTB, GAPDH and RPL13A genes as well as to promote cell survival in response to cisplatin-induced DNA damage suggests the complexity of the impact of CSB somatic mutations. Future studies are needed to catalog the effect of CSB somatic mutations on its function, which would be expected to aid cancer diagnosis and treatment.CSB is a multifunctional protein that participates in a number of nuclear processes, including transcription regulation ,8, UV reMammalian expression constructs of Myc-tagged CSB and CSB-ΔWHD have been described ,17. WildAntibodies used include CSB/ERCC6 ; CSB ; anti-Myc ; RNAPII ; γ-tubulin .All cells were grown in DMEM medium with 10% fetal bovine serum supplemented with non-essential amino acids, L-glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin. Cell lines used: Phoenix , U2OS 4 (ATCC), 2O. DNA was then analyzed by qPCR using previously described primer pair sets [ChIP assays were carried out as described . For eacair sets  (also shImmunoblotting was performed using whole cell extracts as described . BrieflyClonogenic survival assays were done as described . Twenty-


Several antibiotics targeting the large ribosomal subunit interfere with translation in a context-specific manner, preventing ribosomes from polymerizing specific amino acid sequences. Here, we reveal kasugamycin as a small ribosomal subunit-targeting antibiotic whose action depends on the sequence context of the untranslated messenger RNA (mRNA) segments. We show that kasugamycin-induced ribosomal arrest at the start codons of the genes and the resulting inhibition of gene expression depend on the nature of the mRNA nucleotide immediately preceding the start codon and on the proximity of the stop codon of the upstream cistron. Our findings underlie the importance of mRNA context for the action of protein synthesis inhibitors and might help to guide the development of better antibiotics. Escherichia coli proteins. Differential action of KSG significantly depends on the nature of the mRNA residue immediately preceding the start codon, with guanine in this position being the most conducive to inhibition by the drug. In addition, the activity of KSG is attenuated by translational coupling as genes whose start codons overlap with the coding regions or the stop codons of the upstream cistrons tend to be less susceptible to drug-mediated inhibition. Altogether, our findings reveal KSG as an example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action.Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (tRNA) to the small ribosomal subunit, thereby preventing initiation of protein synthesis. Here, by using genome-wide approaches, we show that KSG can interfere with translation even after the formation of the 70S initiation complex on mRNA, as the extent of KSG-mediated translation inhibition correlates with increased occupancy of start codons by 70S ribosomes. Even at saturating concentrations, KSG does not completely abolish translation, allowing for continuing expression of some  The recognition of RBS is facilitated by favorable mRNA folding and modulated by the interaction between a purine-rich Shine-Dalgarno sequence found upstream of the start codon of many genes and a complementary sequence at the 3′ end of the 16S ribosomal RNA , produced by scovered   of the drug was found to result in selective synthesis of some proteins possibly due to stress-induced shortening of 5′ untranslated regions (UTRs) of their mRNAs and also generation of aberrant ribosomes  failed to completely abolish translation, which continued at ∼10% of that in untreated cells even after a prolonged exposure to the antibiotic  gel electrophoresis. The radiograms of the gels exposed for the same amount of time showed the presence of distinct radioactive spots representing a subset of polypeptides actively expressed in the KSG-treated cells . Other polypeptides, actively translated in the untreated cells, were not radiolabeled in the KSG-treated cells .To obtain a more detailed view of the effect of KSG upon translation of a broad array of bacterial genes, we used ribosome profiling (Ribo-seq), a technique that employs deep sequencing of the ribosome-protected mRNA fragments (ribosomes footprints [rfps]) to monitor the progression of translating ribosomes along mRNA , 37. In  E. coli , which athe drug , we wereSI Appendix, Fig. S2B). While challenging the conventional view on the mode of KSG action, these Ribo-seq results support the conclusion of our proteomics studies . To account for these effects when assessing gene-specific action of KSG, we analyzed drug-induced changes in translation efficiency (TE), which is calculated as a ratio between the number of Ribo-seq footprints and RNA sequencing (RNA-seq) reads within protein coding sequences. TE reflects the relative number of ribosomes translating individual mRNA cistrons, and its change reveals altered translation of mRNA independent of its abundance , and the inhibition became more pronounced at 10 mg/mL  of the drug (Top). However, the magnitude of the antibiotic effects varied significantly between individual genes, leading to the significant dispersion of the data points in the TE plots . Consistent with the preferential presence of G(−1) in the most affected genes . pLogo analysis of the ORFs most significantly affected by the drug  C and D sed genes C and D, ed genes E and F. cspE, which carries G(−1) and whose in vivo translation was nearly abolished at 10 mg/mL of KSG (hha with U(−1), whose translation in the presence of the drug continued at a considerable level compared to the untreated control (cspE with the native G(−1) was readily inhibited by KSG, as very few ribosomes reached the Ile4 trap codon when KSG was present in the reaction  or the A(−1) or C(−1) mutations was only modestly affected by KSG, while mutating U(−1) to G significantly sensitized hha translation to the antibiotic inhibition, as judged from the disappearance of the toeprint bands at the “hungry” Pro5 codon  and in vitro (toeprinting) data strongly argue that the identity of the mRNA nucleotide immediately upstream of the start codon greatly influences the inhibitory action of KSG; G(−1) emerged as a key determinant of susceptibility to KSG.The conventional view of the mechanism of KSG action presumes that the drug inhibits binding of canonical (leadered) mRNAs to the small ribosomal subunit, thus interfering with formation of the 30S IC and, consequently, preventing assembly of the 70S IC at the start codon. However, in KSG-treated cells, we observed a dramatic increase of the relative start codon occupancy by 70S ribosomes across the genome A. The hiTo quantify the gene-specific change in ribosome occupancy of the start codons, we implemented the “head enrichment” metric, which measures the relative ribosome occupancy at the head region compared to that across the body of the ORF B. KSG trTranslational coupling, which is defined as the interdependence of translation of adjacent cistrons on the same polycistronic mRNA, may be mediated by direct recruitment of 30S subunit or 70S ribosomes from the upstream genes to the start codon of the downstream genes , 42, 43.Genome-wide approaches enabled us to gain important insights into the mechanism of action of KSG in the living cell. Our main findings can be summarized as follows: 1) inhibition of translation by KSG is generally associated with increased relative ribosome occupancy near the start codons of the ORFs; 2) KSG inhibits protein synthesis in a context-dependent manner, differentially affecting translation of diverse genes; 3) the nature of the mRNA residue immediately preceding the start codon significantly influences KSG action, with G being the most conducive to inhibition of translation, followed distantly by C and then by U and A; 4) translational coupling, diminishes the inhibitory effect of KSG, likely because of the difference between the de novo initiation and reinitiation pathways.iMet into the E site. The lack of E-site tRNA in the initiating ribosome could explain why the antibiotic exerts its inhibitory action preferentially at the start codons. The increase in the start codon occupancy observed in our Ribo-seq experiments is compatible with either scenario because this technique does not distinguish between the rfps originating from 70S IC or elongation-competent 70S ribosomes. Interestingly, toeprinting did not show ribosome stalling at the start codons . This discrepancy might stem from significant differences in the kinetics of the various initiation steps in vitro versus in vivo  or from the absence in the reconstituted cell-free translation system of cellular factors required for KSG-mediated ribosome arrest.Previously proposed models of KSG action presumed that the drug prevents association of mRNA with the small ribosomal subunit, thereby inhibiting translation primarily at the stage preceding the formation of the 70S IC , 20, 21.It is likely that in the living cell, some of the ribosomes stalled by KSG at the start codons of the ORFs never progress to translate the encoded protein and eventually dissociate from mRNA. However, a significant fraction of ribosomes can occasionally escape from the trap and engage in productive translation even at a very high concentration of KSG . The peculiar distribution of the rfp density within the first several codons of the genes revealed by the metagene analysis A suggestSI Appendix, Fig. S5). Clearly, rearrangements of the mRNA trajectory or repositioning of the drug would be required to simultaneously accommodate mRNA and KSG within the mRNA channel. KSG bound in the putative second site, as was observed in the T. thermophilus 30S subunit  trend may operate at the level of formation of the 30S IC or at the later steps preceding translation elongation. While our data provide little insights into the action of the drug prior to 70S IC formation, we observed a more pronounced accumulation of 70S ribosomes at the start codons of the genes with a G(−1) D and E.  subunit , would a subunit . An impo subunit –46, but An additional layer of KSG selectivity stems from translational coupling. Expanding the previous general observations that translation of downstream genes in polycistronic operons tend to be less affected by KSG , our Rib47E. coli genome . Our data strongly argue that gene selectivity of KSG action is manifested with the canonical mRNAs.Some of the earlier studies pointed to a contrasting effect of KSG on translation of the leadered  and phage-encoded leaderless mRNAs, with the latter ones being less affected by the antibiotic , 25, 26.i genome , 35 did oli ORFs A and B. nscripts  as well nscripts . It is png mRNAs : while wOur findings allow us to propose the general model of KSG action which synthesizes our results and the previous findings . AccordiSI Appendix, Supplementary Materials and Methods.The inhibition of protein synthesis by KSG was analyzed by metabolic labeling as described previously , with thE. coli protein was isolated from the 50 mL exponentially growing E. coli culture (strain MG1655) as described previously  and quenched after 3 min with an excess of unlabeled L-methionine.Total eviously . The expSI Appendix, Supplementary Materials and Methods for experimental details.The isolated proteins were resolved following the 2-D Electrophoresis Workflow manual (fourth edition) (BioRad). See SI Appendix, Supplementary Materials and Methods. Total RNA for the RNA-seq analysis was phenol extracted from the same lysate that was used for Ribo-seq. Short RNA and ribosomal RNA were removed from the total RNA with MEGAclear transcription clean-up kit  and MICROBExpress bacterial mRNA enrichment kit , respectively. RNA was fragmented using RNA Fragmentation Reagents  by incubating at 95 °C for 1 min 45 s. The RNA fragments were separated in Novex 15% TBE-Urea gel . The fragments in the 25 to 45 nt range were excised and converted to the sequencing library using the same strategy as for Ribo-seq .Ribo-seq was performed following the procedure described in Ref. http://hannonlab.cshl.edu/fastx_toolkit/). The sequences were then mapped to the reference E. coli MG1655 genome (NC_000913.2.fna) obtained from the National Center for Biotechnology Information (NCBI) Reference Sequence Bank using Bowtie version 1.2.3 (http://bowtie-bio.sourceforge.net/index.shtml), allowing for no more than two mismatches. Reads mapped to more than one locus were discarded. Ribosome density was assigned to the 3′ end of the reads and then adjusted by a shift of −12 nt so it reflects the location of the first base of A-site codon. RNA-seq reads were similarly mapped to E. coli MG1655 genome. Uniquely mapped reads were equally assigned to all the bases that each read covers. For example, for a RNA-seq read that is N-nt long, all the bases covered by the read were given a score of 1/N.Raw reads were filtered for quality and the linker sequence was removed using FASTX-Toolkit  were generated by PCR as described in SI Appendix, Supplementary Materials and Methods using respective primers listed in SI Appendix, Table S4. Toeprinting analysis was carried out as described previously . When needed, the Pro-RS inhibitor 5′-O-[N-(L-prolyl)-sulfamoyl] adenosine Supplementary File


Despite a major interest in understanding how the endothelial cell phenotype is established, the underlying molecular basis of this process is not yet fully understood. We have previously reported the generation of induced pluripotent stem cells (iPS) from human umbilical vein endothelial cells and differentiation of the resulting HiPS back to endothelial cells (Ec-Diff), as well as neural (Nn-Diff) cell lineage that contained both neurons and astrocytes. Furthermore, the identities of these cell lineages were established by gene array analysis. Here, we explored the same arrays to gain insight into the gene alteration processes that accompany the establishment of endothelial vs. non-endothelial neural cell phenotypes. We compared the expression of genes that code for transcription factors and epigenetic regulators when HiPS is differentiated into these endothelial and non-endothelial lineages. Our in silico analyses have identified cohorts of genes that are similarly up- or downregulated in both lineages, as well as those that exhibit lineage-specific alterations. Based on these results, we propose that genes that are similarly altered in both lineages participate in priming the stem cell for differentiation in a lineage-independent manner, whereas those that are differentially altered in endothelial compared to neural cells participate in a lineage-specific differentiation process. Specific GATA family members and their cofactors and epigenetic regulators  with a major role in regulating DNA methylation were among participants in priming HiPS for lineage-independent differentiation. In addition, we identified distinct cohorts of transcription factors and epigenetic regulators whose alterations correlated specifically with the establishment of endothelial vs. non-endothelial neural lineages. Cellular differentiation is fundamental in the development of multicellular organisms and has long been a subject of intense investigation. With current advancements in gene and cell therapy, a clear understanding of the cellular processes that are evoked to establish a specific cell phenotype are central towards designing appropriate cellular manipulations for therapeutic purposes. Among various cell types, endothelial and neural cells are highly attractive targets for therapeutic manipulation, due to their involvement in a multitude of prevalent vascular and nervous system disorders. Dysfunction of endothelial cells that cover the lumen of all blood vessels is a major contributing factor to many cardiovascular, peripheral vascular and cerebrovascular diseases, and many diseases of the central nervous system involve damage to neurons/neural cells. Exploration of the mechanism of endothelial cell specific gene regulation, as well as gene knock out animal models and stem cell differentiation approaches have led to identification of a number of transcriptional regulators that have emerged as major participants in the establishment of the endothelial cell phenotype ,2,3,4. TThe generation and manipulation of stem cells, especially induced pluripotent stem (iPS) cells, combined with high throughput gene expression and computational biology advancements, have provided unprecedented opportunities to design and implement in vitro experimental approaches towards addressing these questions. These approaches have been successfully used to generate a wealth of information regarding changes in gene expression patterns that accompany the establishment of specific cell phenotypes, including endothelial and neural cells ,16. HoweA potential approach towards addressing this gap in knowledge is to compare the molecular signature of two distinct cell lineages that are derived from one parental stem cell, thus possessing a similar genetic background with each other as well as with the parental stem cell. We have previously reported the generation of induced pluripotent stem cells (iPS) from human umbilical vein endothelial cells (HUVEC) the and subsequent differentiation of the resulting iPS (referred to as HiPS) back into a highly pure population of endothelial cells (Ec-Diff), as well as a population of neural cells that contained both neurons and astrocytes (Nn-Diff) ,18. AlthA comparison of HUVEC, HiPS and EC-Diff transcriptome by gene array provided information regarding transcription regulatory factors, including chromatin and DNA modifiers, as well as cofactors as potential contributors to the establishment of endothelial cell phenotype . FurtherBased on the criteria described in Methods, genes that were significantly altered in HiPS-derived endothelial (EC-Diff) or neural (Nn-Diff) cells (relative to HiPS) were identified and a scatterplot of differentially expressed genes generated. To gain insight into the nature of the genes that were altered, they were sorted according to gene ontology, generating a hierarchical structure that is based on their functional characteristics . Genes wFor both endothelial and neural lineages, signaling was the largest group of genes with significant changes, followed by transcription factors and adhesion subsets . The lowWe propose that transcription factors that are upregulated in both lineages  participFurthermore, we suggest that transcription factors, which were downregulated in both lineages , are inhThe coregulation of GATA 3 and 6 transacting factors, together with their regulators ZFPM2 (FOG2) and HEY2, may suggest their similar contribution towards differentiation of both neuronal and endothelial lineages. Among the genes upregulated in both lineages (NR2F2 (COUPTFII), MEF2C, HOXB2, HOXA4 and RARB), NR2F2 (COUPTFII) and MEF2C have previously identified roles in both endothelial and neuronal lineage specification ,25. HOXBWe propose that transcription factors that are specifically upregulated in endothelial cells but remained unchanged in neuronal cells are those that actively participate in establishing endothelial cell phenotype . The lisNotably, the top 10 most highly upregulated transcription factors in this group were ERG, FLI1, TAL1, EPAS, MECOM, TFEC, HOPX, SOX17, SOX7 and SOX18. Among this group, ERG and FLI1 are the members of the ETS family of transcription factors with well-established endothelial-specific gene regulatory function . TAL1 haMECOM is a transcription factor that, in addition to DNA-binding activity, exhibits chromatin-modifying functions ,47. It iThe SOX family of transcription factors is known to participate in cell fate determination, including maintenance of stem cell phenotype . Indeed,https://www.genecards.org/cgi-bin/carddisp.pl?gene=ZNF90, accessed on 23 April 2021).The establishment of endothelial cell phenotype may also be associated with the repression of specific transcription factors, which if present would interfere with the process. While low levels of some transcription factors may indicate no significant change during endothelial differentiation, others may be actively repressed, which could suggest a requirement for their specific downregulation to promote endothelial differentiation. Analyses of transacting factors that are specifically downregulated in endothelial cells  will proThe results presented here suggest the establishment of endothelial cell phenotype as being associated with the upregulation of specific SOX, ETS and GATA transcription factor family members, TFEC and HOXP that participate in hemato-endothelial progenitor establishment, EPAS1 that is responsive to oxygen levels and transcription factor MECOM with multiple chromatin modification functions. It is noteworthy that a number of these transcription factors have an expression pattern that is not restricted to endothelial cells, suggesting a potential combinatorial role for this cohort of transacting factors in establishing endothelial cell phenotype. Additionally, of interest is the GATA transcription factor family, which includes members such as GATA2 that participate in endothelial lineage establishment, and others such as GATA3 and 6 that are associated with the lineage-independent differentiation process.Among the factors that were downregulated in endothelial cells, two were of special interest since they were specifically upregulated in neuronal cells , thus suBased on similar reasoning to that used for endothelial cells, we propose that genes that are specifically upregulated and downregulated in neural cells are those that participate in the establishment of neural cells phenotypes. In the category of the genes that were upregulated in neural cells , the majAs discussed for endothelial cells, the establishment of neural lineage may also require downregulation of certain transcription factors. Seven transcription factors, including FOXO1, MEOX2, ZSCAN10, NFE2L3, HMX2, ARNTL2 and TCF19, were specifically downregulated in neural cells . FOXO1, Another significantly downregulated transcription factor was MEOX2, which has a major role in vascular biology . MEOX2 iAs expected, genes in this category not only included factors that are reported to participate in the maintenance of pluripotency, such as ZSCAN10  and NFE2Overall, these results suggest that differentiation towards neural phenotype is concomitant with the downregulation of genes that regulate: (i) specific cellular metabolisms, (ii) circadian cycles, (iii) response to external cues that could trigger apoptosis, (iv) vascular cell specification and (v) maintenance of stem cell phenotype.We also identified a number of transcription factors that were upregulated in one lineage and downregulated in the other. Identification of these factors provides a novel insight towards dual functioning transacting factors that may function as an activator of genes required for establishing one lineage while simultaneously repressing genes associated with establishment of a different lineage. We did not identify any transcription factors that were specifically upregulated in endothelial cells while being downregulated in neural cell lineage. However, we did identify two transcription factors, IRX2 and IRX3, that were upregulated in neural and significantly downregulated in endothelial cells . IRX2 anIRX2 was shown to participate in cerebellum formation and potentially the ventricular conduction system (VCS) ,90, whilEpigenetic modification of genes is another process that significantly contributes to activation/repression of gene expression . As discDNMT3B, PRDM14 and HELLS were identified as epigenetic regulators that were downregulated in both endothelial and neuronal lineages . DNMT3B PRDM14 (a member of the PRDM family of epigenetic regulators with histone methyl transferase activity ) is a DNGenes coding for epigenetic factors that were significantly altered in endothelial cells included three genes  that were upregulated  and eighPCGF5 is a histone modifier (mediates ubiquitylation of histone H2A) which exerts both repressive and activating functions and was reported to participate in neuronal differentiation . Thus, iIn addition to alteration in DNA methylation, histone modifications and chromatin remodeling, epigenetic regulation of gene expression may also encompass regulation of mRNA levels. This may involve the function of non-coding RNAs (such as miRNA and lncRNA) or RNA binding proteins that regulate target mRNA levels (including mRNAs coding for epigenetic regulators as well as transcription factors) ,112.Of the eight genes in the epigenetic category that were downregulated , LIN28A,TET1 is a member of TET (ten–eleven translocation) family of dioxygenase enzymes that participate in DNA demethylation at the 5-methylcytosine (5mC) base, and thus contributes to an epigenetic modification generally consistent with transcriptional activation . HoweverThe cohort of genes with epigenetic function that were altered in neural cells included two genes (BCORL1 and LHX2) that were upregulated  and one Overall, epigenetic regulators, although generally lacking inherent specificity, exhibit differential expression patterns based on cellular phenotype and may exert lineage-specific effects based on their level of expression. Additional measures of specificity could be added through association of many epigenetic regulators with DNA binding transcription factors that recruit them to the target promoters. Recently, distinct RNA binding proteins have been identified as major epigenetic regulators, which exhibit lineage specificity and could present novel targets for exploring the epigenetic regulation of cell fate outside the chromatin structure ,135,136.Collectively, based on the results of the gene array analyses, we posit that the establishment of a distinct cell fate from pluripotency involves progression through stage(s) that independent of the final phenotype destination, poises the cell for differentiation. This process involves alterations in gene expression pattern compared to the pluripotency state and is independent of the final-destination phenotype. Once a cell is poised to differentiate, establishment of the final-destination phenotype  involves additional alterations in gene expression pattern that are lineage specific. Although these alterations include genes belonging to multiple functional groups, those involved in transcription regulation, including transcription factors and epigenetic regulators, are uniquely positioned to alter activities of multiple networks of target genes as master regulators. Thus, we focused on these two ontology groups and propose the following model  to descrWe hypothesized that transcription factor and epigenetic genes that are commonly up- or downregulated in both endothelial and neural lineages potentially promote lineage-independent pro-differentiation phenotypes. However, these analyses will not allow distinguishing among cells that may be poised to differentiation towards all, multiple, or a select few phenotypes. Furthermore, since cells differentiated into neural lineage constituted both neurons and astrocytes, the gene array analyses from these cells’ populations do not provide information regarding the neuronal- vs. astrocyte-specific gene expression profile. However, considering that both neurons and astrocytes belong to neural lineages, it remains informative with regard to exploring alteration in gene expression that occurs when iPS are differentiated towards endothelial compared to a distinct non-endothelial neural lineage. Notwithstanding the caveats, we propose that being poised to differentiate is associated with the downregulation of three epigenetic regulators  with major roles in regulating the DNA methylation profile. Since two of the three (DNMT3B and HELLS) are associated with de novo methylation, while PRDM14 has a demethylation function, their downregulation may alter the epigenetic landscape of a stem cell to prime lineage-specific genes for activation, while rendering stem cell-specific genes poised for repression. These observations highlight alterations in DNA methylation patterns as a major epigenetic event in priming a pluripotent cell for differentiation. Additionally, this process appears to be strongly associated with a major increase in GATA transcription factor activity, since an increase in the gene expression of select GATA family members  as well as GATA cofactor (ZFPM2), in combination with the downregulation of GATA repressor (HEY2), is prominent. The establishment of neural- or endothelial-specific lineages then requires additional gene expression alterations  that are restricted to the target lineage. While these results require experimental confirmation, it provides a potential list of target genes that could be prioritized for experimental approaches to determine their role individually and/or collectively in differentiation process.p-value from the fcros’ package was above 0.95 and their mean change in expression was greater than 1.5. Similarly, genes were considered significantly downregulated if their adjusted p-value from fcros’ was below 0.05 and their mean change in expression was less than −1.5.The initial dataset contained 49,400 observations for 19,929 unique genes ,18. Each


Saccharomyces cerevisiae, the main genomewide safeguard against pervasive ncRNAs is the Nrd1-Nab3-Sen1 (NNS) complex, composed of two RNA-binding proteins (Nrd1 and Nab3) and the helicase Sen1. The NNS complex directs transcription termination of ncRNA genes and promotes the rapid degradation of pervasive transcripts from yeast nuclei through its physical and functional coupling to the nuclear RNA exosome. We have recently shown that inhibition of the exosome in yeast cells leads to the accumulation of ncRNAs complexed with Nab3 and Nrd1, decreasing recycling of these termination factors to sites of transcription and inducing global termination defects at NNS targets. Consistent with the notion that ncRNAs out-titrate Nab3 and Nrd1 termination factors, we have shown that a similar genomewide termination impairment could be achieved by expressing a circular RNA decoy containing a Nab3 binding target S.cerevisiae cells, normalized by spiked-in S.pombe lysates. These data allow the useful assessment of the extent of co-transcriptional binding decrease of Nab3 by decoy ncRNA titration and will be valuable for further analyses of NNS targeting mechanisms.Pervasive transcription originating from the ubiquitous activity of RNA Polymerase II (RNAPII) generates a vast mass of non-coding RNAs (ncRNAs) that represent a potential harm to gene expression. In the compact genome of the yeast  They can help conceive experiments to test the reactivity of diverse NNS-terminated RNA groups to different stresses and stimuli.1rrp6Δ cells or by expression of a circular ncRNA decoy  protocol S.pombe lysates containing HTP-tagged RNAPII. After single-end sequencing of cDNA libraries, subsequent raw reads filtering, and mapping to reference yeast genome (R64), the normalized datasets  were analyzed using deeptools 2.0 https://usegalaxy.eu/). Analysis of the correlation among biological replicates revealed significant Pearson correlation coefficients (We have recently shown that the transcription termination control that the Nrd1-Nab3-Sen1 (NNS) complex exerts on pervasive nuclear non-coding RNAs (ncRNAs), ultimately leading to their degradation by the nuclear RNA exosome, can be impaired by the sequestration of termination factors by accumulation of ncRNAs NA decoy . While ificients . Next, rficients A. Then, ficients B, as welficients C. In allficients A and mRNficients B aligned22.1S.cerevisiae strain DLY3402 (MATa NAB3::HTP::TRP1 dbr1∆::HIS3 ura3–1 ade2–1 leu2–3112 can1–100) carrying either the control pTet-i or the decoy pTet-i-CUT constructs  was grown in EMM+5AA medium to OD600 of 0.5 at 30 °C before being subjected to UV crosslinking in vivo and subsequently processed for CRAC.Yeast 2.2S.pombe lysates containing HTP-tagged RNAPII to allow better normalization of the three independent biological replicates analyzed.CRAC was performed exactly as described in the related research paper 2.3http://hannonlab.cshl.edu/fastx_toolkit/) and mapped to the R64 genome S. pombe spike in present in the sample, reads that did not map on S. cerevisiae were mapped on the S. pombe genome after filtering for reads shorter than 40 nucleotides to avoid cross mapping of S. cerevisiae reads on S. pombe. The amount of mapped spike-in reads was used for normalization purposes.CRAC datasets were analyzed as described previously 2.4http://deeptools.ie-freiburg.mpg.de/) feeding bigwig files to deeptools 2.0 2 Decoy/CTRL), features were sorted by decreasing Nab3 average signal within the first 500 nucleotides downstream of the TSS. For correlation plots, the multiBigwigSummary and plotCorrelation Galaxy tools were used and the Pearson correlation coefficient in 1 kb bins was calculated for each comparison.Normalized genomic data obtained from CRAC were analyzed on the Freiburg Galaxy platform (Tommaso Villa: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original draft preparation, Reviewing and Editing; Yan Jaszczyszyn: Methodology; Domenico Libri: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original draft preparation, Reviewing and Editing, Funding acquisition, Supervision.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


Genomic imprinting is an epigenetic phenomenon that results in unequal expression of homologous maternal and paternal alleles. This process is initiated in the germline, and the parental epigenetic memories can be maintained following fertilization and induce further allele-specific transcription and chromatin modifications of single or multiple neighboring genes, known as imprinted genes. To date, more than 260 imprinted genes have been identified in the mouse genome, most of which are controlled by imprinted germline differentially methylated regions (gDMRs) that exhibit parent-of-origin specific DNA methylation, which is considered primary imprint. Recent studies provide evidence that a subset of gDMR-less, placenta-specific imprinted genes is controlled by maternal-derived histone modifications. To further understand DNA methylation-dependent  and -independent  imprints, this review summarizes the loci under the control of each type of imprinting in the mouse and compares them with the respective homologs in other rodents. Understanding epigenetic systems that differ among loci or species may provide new models for exploring genetic regulation and evolutionary divergence. Mus musculus) and Brown Norway rat (Rattus norvegicus) become the second and third mammals to have their genomes sequenced in 2002 and 2004, respectively  plus one set of sex chromosome (X or Y) to the offspring. According to Mendel’s law, diploid cells contain parental copies of each autosomal gene, which are predicted to show the same transcription state. However, “genomic imprinting” is a form of non-Mendelian inheritance that results in parent-of-origin allele-specific gene expression of autosomal loci or of loci on the diploid X chromosome . Polymorphism information between strains or individuals helps distinguish between paternal and maternal alleles. It was only natural that the phenomenon of genomic imprinting was discovered and well-investigated in mice, where nuclear transfer and genetic engineering technologies have always been developed and where numerous strains  have been established and maintained.In 1984, two laboratories published landmark papers that reported a new phenomenon in mammalian genome biology . Both stNevertheless, not all chromosomes produce abnormalities when present as disomies, depending on which chromosome or part is made uniparental; however, those commonly lead to striking phenotypic differences. These investigations revealed the requirement of both maternal and paternal genomes for normal development, which was tied to an intriguing biological phenomenon called genomic imprinting. Uniparental inheritance of the genome or chromosome occurs spontaneously in humans, resulting in early pregnancy losses, like androgenetic and parthenogenetic conceptuses (hydatidiform moles and benign ovarian teratomas), or moderate to severe developmental disabilities, known as imprinting diseases . SubsequTme  was identified on the proximal mouse chromosome 17 overlapping deletions of maternal-effect lethal mutants, like Thp or lub2T  exclusively expressed from the maternal allele, and Igf2 originates from the opposite allele. Interestingly, the opposite imprinting of Igf2 and its scavenging receptor gene, Igf2r, demonstrates conflicting parental effects of growth promotion and growth restriction, which supports the classic “parent-offspring conflict theory” for the evolution of genomic imprinting  is acquired prenatally in prospermatogonia before the onset of meiosis in the male germline (de novo DNA methyltransferase (DNMT) 3a and the accessory protein DNMT3L  pathway and the rodent-specific DNMT3C are also responsible for the establishment of paternal DNA methylation , has been studied as the best candidate for the molecular mechanism of inheriting parental-specific imprints following fertilization. Because parental imprints must be established when the parental genomes can be distinguished, investigators assayed methylation acquisition during gametogenesis, when the maternal and paternal genomes are entirely separated and can be independently epigenetically modified. Paternal-specific methylation of the gDMRs at three imprinted loci , Smoc1, and Slc38a4, in mice  originating from an upstream transcription start site, which is regulated by a maternal gDMR  and 16 maternal  gDMRs were conserved between mice and humans  that undergo paternal methylation imprinting in mice  genes within intron 10, and these miRNAs are also imprinted and essential for placental development  drives its placental role in rodents.It has already been shown that single or multiple genes are imprinted on the homologous regions of the three imprinted clusters  result in strong parent-of-origin effects on placental and embryonic growth  and maternally (such as H19) expressed genes were also reidentified in hamster. Smoc1 encodes a multi-domain secreted protein that may play a critical role in ocular and limb development (Smoc1 is not likely associated with loss-of-imprinting in hybrid hamsters, and its functional role in placental development remains unknown.In hamsters, reciprocal crosses between two dwarf hamsters (c growth . The expc growth . Single-pression . While ielopment . HoweverThe discovery of a non-canonical imprinting mechanism mediated by histone modifications is an important finding that provides a new molecular mechanism for epigenetic transgenerational inheritance. In contrast, the diversity of canonical and non-canonical imprinting complicates our understanding of the underlying mechanisms and a better understanding of the differences among mammalian species that bridge the gap between humans and mice. For instance, the insertion of endogenous retroviral elements drives both canonical and non-canonical imprinting . 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Yin Yang 1 (YY1) which are mainly involved in cell proliferation, biological adhesion, and metabolic processes. We found here that the ChIP-Seq peaks of MOF and NSL3 co-localized with H4K16ac, H3K4me2, and H3K4me3 at the transcriptional start site of YY1. In addition, both the mRNA and protein expression levels of YY1 were regulated by silencing or overexpressing NSL HAT. Interestingly, the expression levels of cell division cycle 6, a downstream target gene of YY1, were regulated by MOF or NSL3. In addition, the suppressed clonogenic ability of HepG2 cells caused by siNSL3 was reversed by overexpressing YY1, suggesting the involvement of YY1 in NSL HAT functioning. Additionally, de novo motif analysis of MOF and NSL3 targets indicated that the NSL HAT complex may recognize the specific DNA-binding sites in the promoter region of target genes in order to regulate their transcription.The human males absent on the first (MOF)-containing non-specific lethal (NSL) histone acetyltransferase (HAT) complex acetylates histone H4 at lysine K5, K8, and K16. This complex shares several subunits with other epigenetic regulatory enzymes, which highlights the complexity of its intracellular function. However, the effect of the NSL HAT complex on the genome and target genes in human cells is still unclear. By using a CRISPR/Cas9-mediated NSL3-knockout 293T cell line and chromatin immunoprecipitation-sequencing (ChIP-Seq) approaches, we identified more than 100 genes as NSL HAT transcriptional targets, including several transcription factors, such as  Dynamic changes in chromatin structure are mainly regulated by epigenetic regulatory enzymes, such as ATP-dependent chromatin remodeling enzymes, histone variant exchange enzymes, and post-translational histone-modifying enzymes . In cellDrosophila, MSL HAT plays a critical role in the regulatory mechanism of X-chromosome dosage compensation [Highly dynamic intracellular histone acetylation is controlled by histone acetyltransferases (HATs) and histone deacetylases . Human mensation ,6. Howevensation ,8. Basedensation ,12,13,14O-linked N-acetylglucosamine (OGT1), WD-40 repeat protein 5 (WDR5), host cell factor 1 (HCF1), microspherule protein 1 (MCRS1), and plant homeodomain finger-containing protein 20 (PHF20), has a broader substrate specificity, and can simultaneously acetylate histone H4K16, H4K5, and H4K8 [ANKRD2) [O-GlcNAc-modified site. Moreover, NSL HAT, but not MSL, is essential for cell survival [Compared with the MSL, the NSL HAT complex consists of nine subunits, including MOF, NSL1-3, and H4K8 , indicat[ANKRD2) . Further[ANKRD2) . NSL3 issurvival ,20. Takesurvival ,22.Drosophila, NSL HAT is positioned at +1 in the nucleosome at NSL-bound gene promoters, which is pivotal for not only effective transcription but also transcriptional start site (TSS) fidelity [Drosophila chromatin-remodeling enzyme imitation switch to the chromatin fiber [Recently, increasing evidence regarding the role of the NSL HAT complex in the genome has been published. For example, the NSL HAT complex primarily presents at active promoters of housekeeping genes . In Drosfidelity . It is afidelity . It shoufidelity ,27. Addiin fiber , suggestin fiber . In cellin fiber . For exain fiber . The curin fiber . Enhancein fiber ,34. Enhain fiber . When enin fiber . In addiin fiber . DespiteNetworks between transcription factors (TFs) and histone-modifying complexes can indirectly affect histone marker-mediated gene transcription. TFs recruit histone modifiers to the promoters or enhancers of their target genes and establish an altered structure for the transcription of those genes . For exaMOF, as a catalytic subunit, forms both MSL and NSL—two distinct multiprotein complexes A. Both cThe hallmarks of cancer include sustained chronic proliferation and elevated proliferative signaling . AccordiYY1 was particularly conspicuous. It is well known that YY1 is upregulated in various types of tumors and is crucial for tumor cell proliferation and metastasis [To clarify the role of the NSL HAT complex in cell proliferation, genes whose promoter regions (TSS ± 200 bp) overlapped with the ChIP-Seq peaks of MOF/MYST1 or NSL3 were further analyzed. Sixteen co-bound genes were screened E, among tastasis . Figure YY1 mRNA levels in 293T or HeLa cells  targeted exon 2, which prevented NSL3 expression A,B. GlobNSL3-KO resulted in a significant decrease in the occupancy of MOF/MYST1 at NSL3-bound sites, indicating the function of NSL HAT in genome stability. Thus, to clarify how NSL3-KO-mediated redistribution of the NSL HAT complex within the genome affects gene expression, ChIP-Seq using antibodies against Pol II, H4K16ac/K5ac/K8ac, H3K4me1/me2/me3, H3K27ac, and H3K27me3 was performed. NSL3-KO did not lead to changes in the level of total Pol II at the TSSs of all genes , and distal intergenic region. As shown in To further understand the genome-wide binding of histone modifications and Pol II, Pearson correlation coefficients were calculated to indicate the degree of concurrence of histone marks and Pol II at all gene regions. The NSL HAT complex corresponding to histone H4 acetylation, including H4K16ac/K5ac/K8ac, was positively associated with the transcriptional marks of Pol II and H3K4me2/me3 in NSL3-WT 293T cells. Although the aforementioned correlation coefficients changed in the order of strength in NSL3-KO cells, H4K16ac, H4K8ac, and H4K5ac still maintained a positive relationship with the transcriptional marks of Pol II and H3K4me2/me3, suggesting that the crosstalk between histone H4 acetylation and H3K4 methylation was associated with gene transcription. In addition, NSL3-KO seemed to have a minimal effect on the enhancer marks of H3K27ac and repressor marks of H3K27me3, but H3K4me1 was greatly affected by NSL3-KO, indicating that NSL3-KO may facilitate the deposition of the H3K4me1 mark within the enhancer region A. Based In order to identify genes and biological pathways regulated by NSL HAT through histone modifications, we further characterized genes that coordinated modifications at H4K16ac, H3K4me2, and H3K4me3 orchestrate the NSL HAT mediated transcription. First, the genes bound together by H4K16ac, H3K4me2, and H3K4me3, which are also present in cluster 8 or cluster 9, were selected E. To betKLF6, MED30, FOXP2, TAF15, XBP1, solute carrier family 3 member 2 (SLC3A2), ubiquitin like modifier activating enzyme 2 (UBA2), ubiquitin C-Terminal hydrolase L5 (UCHL5), nicotinamide phosphoribosyl transferase (NAMPT), and dyskerin pseudouridine synthase 1 (DKC1) was significantly downregulated in NSL3-silenced HeLa cells , TATA-binding protein associated factor 15 (TAF15), and mediator subunit 30 (MED30); however, this phenomenon was suppressed by NSL3-KO A. To furLa cells C.Given that the NSL HAT complex forms a gene regulatory network with multiple TFs, we speculated that the NSL HAT, as a transcriptional cofactor, may participate in the transcriptional regulation of certain genes. Thus, to address this speculation, we performed de novo motif analysis for MOF and NSL3 ChIP-Seq data using HOMER in 293T cells. The promoter-associated motifs were defined via NSL HAT binding sites within TSSs ± 200 bp. The analyzed binding sequence results showed that the common promoter binding sequences derived from MOF and NSL3 ChIP-Seq matched the DNA-binding motifs of several known TFs, such as KLFs, E74 Like Factors (ELFs), and YY1, suggesting that these factors may cooperate with the NSL HAT complex to regulate gene transcription D. Based MOF, a member of the MYST HATs, is involved in many biological processes through specifically acetylating histone H4K16 ,27. In cThe NSL3, a key subunit of the NSL HAT complex, plays a crucial role in maintaining the enzymatic activity of the complex through its O-GlcNAc-modification at Thr755 site by OGT1 . In lineANKRD2-activated gene, has been observed [YY1 appeared in co-bound genes. High expression of YY1 is frequently observed in various cancers, including breast cancer, pancreatic cancer, colon cancer, and lung cancer [YY1 transcription , anti-H4K5ac (07-327), anti-H4K8ac (07-328) and anti-H3K4me2 (07-030) polyclonal antibodies were from Merck Millipore . Anti-H3K4me3 (ab8580), anti-H3K4me1 (ab8895), anti-H3K27ac (ab6002) and anti-H3K27me3 (ab4729) polyclonal antibodies were from Abcam . Anti-histone H4 (16047-1-AP) polyclonal antibody was obtained from Proteintech Group . Anti-H3 was obtained from Ruiying Biological . Anti-YY1 (H414) rabbit polyclonal antibody and anti-CDC6 monoclonal antibody (sc-13136) were from Santa Cruz Biotechnology . Anti-Pol II (39097) monoclonal antibody was from Active Motif . Anti-MOF (A300-992A) polyclonal antibody was from Bethyl Laboratories, Inc. . Anti-NSL1, anti-NSL2, anti-NSL3, anti-MCRS1, anti-WDR5, anti-OGT1, anti-PHF20, anti-MYST1 and anti-GAPDH rabbit polyclonal antibodies were raised against bacterially expressed proteins . In addition, the total IgG from NSL3- and MYST1-antisera was purified by affinity chromatography.2 in Dulbecco’s modified Eagle’s medium , and cultured with 10% fetal bovine serum  and 1% penicillin-streptomycin mixture .HEK293T, HepG2 and HeLa cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences  and cells were maintained at 37 °C and 5% COThe coding region of full-length human MOF (BC037773), NSL3 (NM_001115016) and YY1 (NM_003403) with Flag tag were cloned into pcDNA3.1 (−). The flag-tagged full-length MOF, NSL3 and YY1 plasmids were transiently transfected into cells seeded in 6-well plates using polyethyleneimine (PEI) .HeLa or 293T cells were transfected with non-targeting (NT) (D-001206), NSL1 (D-031748) and NSL3 siRNA (M-016928) SMART pool . Cells were transfected with siRNA using Lipofectamine RNAiMAX . The pLVX-shRNA system was used to express shRNA in HepG2 cells, and the target sequences specific for NSL3 were used: shNSL3-1, CCCAGGAAACTTGTGGCATTA; shNSL3-2, GAGCTGATGACAATCTCAGAA; shNSL3-3; CCCACACAAACCCATTATCTT.The second exon sequences of NSL3 region were used for single-guide RNA (sgRNA) target screening. The designed sequence, CAGACTTCAGCTCGACGCAT was ligated into pX459 vector, which expresses Cas9 behind an CMV promoter. We transfected 293T cells with the pX549-NSL3-KO plasmid for 48 h, and clones were selected with 2 μg/mL puromycin. Individual colonies were isolated into the wells of a 96-well plate, and the efficiency of the NSL3-KO transfection was measured by western blotting and genomic DNA sequencing.®Premix Ex Taq™II  and Eco Real-Time PCR System . The PCR reactions were conducted with the manufacturer’s protocol. The following primer sequences were used for RT-qPCR analysis . cDNA synthesis was performed with a PrimeScript 1st Strand cDNA synthesis Kit . Relative mRNA level was measured using TB Greenanalysis :NSL3 wild type (WT) or NSL3-KO 293T cells were cultured to approximately 30% confluence in 24-well plates containing cover slips  in each well. Then, 48 h later, the cells were immunostained according to a method previously described . H4K16acpcDNA3.1 (-) or Flag-NSL3 plasmid-transfected HeLa or HepG2 cells (2000 cells/well) were grown in 96-well plates and incubated overnight. MTT assays were carried out using MTT -2,5-diphenyltetrazolium bromide) reagent  at 1–7-day time points. The spectrophotometric absorbance was measured at 490 nm using a microplate reader .HeLa or HepG2 cells grown to approximately 30% confluence in 6-well plates were transfected with Flag-NSL3 for 48 h. The cells were then split into a new 6-well plate at 2000 cells/well. One week later, the cells were fixed with 4% paraformaldehyde at 37 °C for 15 min, and formed colonies were stained with crystal violet for 1–2 min. The colonies were counted using a MOTIC microscope .A mixture of 0.75 mL DMEM culture medium and 0.75 mL 0.6% low-melting agarose  was added to 6-well plates. Upon solidification, a mixture of 0.75 mL DMEM culture medium, 0.75 mL 0.35% low-melting agarose, and 2000 HepG2 cells/well was added to the top of the solidified layer. The plates were placed at 37 °C for 2 weeks. The clones were observed and captured using an MOTIC microscope. Finally, all the clones were stained with MTT at 37 °C for 4 h and visualized with a digital camera.The promoter region of YY1 (−97 bp to +203 bp) and CDC6 (−1066–+240 bp) was introduced into the pGL3-Luc Vector (Promega Corporation). The luciferase reporter assay was conducted as described previously . Brieflyhttps://www.r-project.org/, accessed on 2 January 2021). Differential expression analysis was performed using DESeq2 [Total RNA from NSL3-WT or NSL3-KO 293T cells was extracted using the RNAiso Plus  and sent to Biomarker Technologies Co., Ltd.  for RNA-Seq analysis. Sequenced reads were aligned to the GRCh38 genome assembly using HISAT2 (v2.1.0) . Counts g DESeq2 . Genes w6 reads per sample.H3K4me1/me2/me3, H4K5ac/K8ac/K16ac, H3K27me3, H3K27ac, Pol II, MOF, MYST1, and NSL3 ChIP assays were performed on NSL3-WT or NSL3-KO 293T cells. Precipitated DNA was extracted with phenol:chloroform:isoamyl (25:24:1) and precipitated with ethanol. ChIP-Seq libraries were prepared using NEBNext Ultra DNA Library Prep Kit  according to the manufacturer’s manual and were then purified with AMPure XP beads . The prepared samples were sequenced using NovaSeq 6000  with a sequencing depth of 30 × 10https://www.r-project.org/, accessed on 2 January 2021).ChIP-Seq reads were trimmed and filtered for quality and adaptors using fastp ; subsequNuclear extract protein from HeLa cells and stored at −80 °C. EMSA probes, NSL3 binding site (CTCCTCGCCCGCCCGCCCGCAGCCG) in YY1 promoter were synthesized and labeled with biotin at 5’-ends of the probes. Unlabeled EMSA probes were made without biotin. The reaction system contains EMSA binding buffer (5×), probes and nuclear extract protein. Anti-NSL3 antibody was added to the reaction system containing biotin labeled probes for super shift assay. After incubation at room temperature for 20 min, the mixture was electrophoresed through a 4% native polyacrylamide gel electrophoresis at 70 V for 1 h, and then the DNA–protein complex was transferred to a positive nylon membrane. DNA was detected using the chemiluminescent EMSA kit  according to the manufacturer’s instructions.t-test. A statistically significant difference was considered to be present at p < 0.05.The data was analyzed using SPSS 16.0 software . The difference between two independent samples was analyzed by Student’s All of the next-generation sequencing data have been deposited to GEO and can be accessed as follows: RNA-Seq (GEO: GSE198646), ChIP-Seq (GEO: GSE198645).