We also found that PARP1 interacts with some MyoD-binding regions and that its presence, independently of the enzymatic activity, interferes with MyoD recruitment and gene induction. We finally suggest a relationship between the binding of PARP1 and the loss of the activating histone modification H3K4me3 at MyoD-binding regions. This work highlights not only a novel player in the epigenetic control of myogenesis, but also a repressive and catalytic-independent mechanisms by which PARP1 regulates transcription.The myogenic factor MyoD regulates skeletal muscle differentiation by interacting with a variety of chromatin-modifying complexes. Although MyoD can induce and maintain chromatin accessibility at its target genes, its binding and trans-activation ability can be limited by some types of not fully characterized epigenetic constraints. In this work we analysed the role of PARP1 in regulating MyoD-dependent gene expression. PARP1 is a chromatin-associated enzyme, playing a well recognized role in DNA repair and that is implicated in transcriptional regulation. PARP1 affects gene expression through multiple mechanisms, often involving the Poly(ADP-ribosyl)ation of chromatin proteins. In line with PARP1 down-regulation during differentiation, we observed that PARP1 depletion boosts the up-regulation of MyoD targets, such as  The entire process is promoted by MyoD, a muscle-specific transcription factor recognized as the master regulator of myogenesis, due to its capability of converting several non-muscle cell types to skeletal muscle cells2. MyoD regulates gene expression through a number of different and interdependent mechanisms, which are being increasingly discovered in the last years4. MyoD is a bHLH protein and interacts with most of its transcriptional targets by recognizing E-box sequences5. The induction of transcription by MyoD relies on its cooperation with other transcription factors, both ubiquitous and muscle-specific, and on its ability to interact with chromatin modifying enzymes1. The best established mechanisms by which the myogenic factor creates a chromatin environment permissive for transcription involves the recruitment of the histone acetylases p300/CBP and pCAF8 and the chromatin remodelling complex SWI/SNF10 on the targeted regulatory-regions. Moreover, MyoD functionally interacts with the arginine methyltransferase Prmt511, with the lysine methyltransferase Set7/912, with the chromatin remodelling enzyme Chd213 and, as more recently reported, with the regulatory Long noncoding RNAs Linc-RAM14 and Kcnq1ot115. MyoD has been shown to modify the chromatin status not only at promoters and enhancers, but also at intergenic regions6. In this regard, using the p57/KvDMR1 locus 16 as a model, we provided evidence that MyoD can regulate gene expression from a distance by releasing repressive chromatin loops19. More recently, a direct role of MyoD in reorganizing the three-dimensional chromatin interactome during the myogenic conversion, has been reported20.Skeletal muscle differentiation is determined by the activation of a complex program of gene expression, which involves the temporally regulated induction of muscle-specific genes as well as of cell cycle inhibitors that ensure cell cycle exit of differentiating cells21. For example, it has been reported that trimethylation of lysine 27 on histone H3 (H3K27me3) at the regulatory regions of certain muscle-specific genes prevents MyoD binding and gene activation in undifferentiated myoblasts. Upon differentiation stimuli, the recruitment of MyoD is enabled by the reduction of H3K27me3 levels, due to the down-regulation of EZH2, the histone methyltransferase that catalyzes this modification22. Similarly, the access of MyoD to a number of differentiation genes, before the onset of differentiation, is blocked by a repressor complex containing Snail and the histone deacetylases I and II, which is removed only upon differentiation stimuli23. The existence of epigenetic barriers for MyoD binding, involving EZH2 and H3K27me3, has also been suggested to contribute to the defective function of the myogenic factor in rhabdomyosarcoma cells24. Moreover, we have recently shown that accumulation of H3 lysine 9 dimethylation (H3K9me2) at a critical regulatory region of the MyoD target p57kip2, hinders the binding of MyoD and the consequent reorganization of the chromatin architecture, thus preventing the up-regulation of the gene in muscle cell types un-responsive for p57 induction17. In light of the complexity of epigenetic regulation of transcription, it is most likely that the molecular mechanisms modulating MyoD binding to chromatin are even more various, an issue that awaits further investigation in relation to both physiological and pathological myogenesis.Although MyoD can initiate chromatin remodeling and histone modifications at target sites in heterochromatin, nevertheless its binding and transactivation capability are limited by some types of epigenetic constraints. In this regard, the poor ability of MyoD to convert some cell types to the muscle lineage has been ascribed, at least in part, to pre-existing chromatin features that preclude MyoD access to its targets26, also termed ADP-ribosyltransferases with diphtheria toxin homology (ARTDs), according to a new nomenclature27. PARPs catalyze the addition of single or multiple ADP-ribose units on target proteins, using NAD+ as a substrate, leading to Mono(ADP-ribosyl)ation or Poly(ADP-ribosyl)ation (PARylation)26. The addition of poly(ADP-ribose) (PAR) polymers is a reversible post-translational modification involved in a variety of cellular processes29. PARP1 is localized predominantly in the nucleus28 and, in part, in mitochondria30 and catalyzes the PARylation of many different types of proteins, among which PARP1 itself, histones, transcription factors and other chromatin proteins31.PARP1 is the most abundant and the best studied family member of the Poly(ADP-ribose) polymerases (PARPs)32. However, there is increasing evidence that PARP1 influences chromatin dynamics and transcription also in response to a variety of signals other than genotoxic stress, such as inflammation, proliferation and differentiation stimuli34. PARP1 has been reported to influence transcription through a variety of molecular mechanisms, with different outcomes on gene expression. It has long been recognized that PARP1 can directly affect the degree of chromatin compaction. The active enzyme induces chromatin decondensation by causing nucleosomal-histone PARylation36 and by displacing the linker histone H1 from chromatin38. On the other hand, inactive PARP1 has been found to function as a structural component of chromatin and to cause chromatin compaction accompanied by transcriptional repression41.The best recognized role of PARP1 is related to the maintenance of genome stability and relies on the modification and recruitment of DNA repair complexes at sites of damaged DNA within chromatin43. For example, active PARP1 promotes histone acetylation at specific promoters44 and supports histone phosphoacetylation at immediate early response genes during the emergency from quiescence45. An additional strategy by which active PARP1 promotes chromatin accessibility, involves the inhibition of EZH2 activity, through PARylation of the histone methyltransferase, and the consequent decrease of the global levels of H3K27me347. Moreover, PARP1-dependent PARylation also inhibits the histone demethylase KDM5B, resulting in the increased trimethylation of lysine 4 on histone H3 (H3K4me3)48, an active chromatin mark. On the other hand, inactive PARP1 was recognized to be a critical component of the corepressor complexes Groucho/TLE and CtBP, which block transcription from the MASH1 and p21 promoters respectively50. According to a simple and elegant model, PARP1 would play a dual role in the transcriptional regulation, involving its participation in gene repression, when inactive, and then in gene de-repression, following the enzyme activation. In other words PARP-1 would function as an exchange factor promoting the switch from repression to activation in response to particular signals50. Some findings, however, do not fit into this simple model. For example, active PARP1 participates in the establishment and maintenance of heterochromatin during X chromosome inactivation and ribosomal DNA silencing, as well as at telomeres and centromeres51. Conversely, inactive PARP1 positively regulates a pluripotency gene expression program in embryonic stem cells52.PARP1 can also indirectly affect the chromatin structure by modulating the pattern of histone modifications and the DNA methylation status54. Some others, such as PPARγ, are instead activated55. As a further strategy of co-regulation, PARP1, independently of its catalytic activity, interacts with and co-activates E2F156 and NFkB57.To complicate the picture, PARP1 functions as a co-regulator of a number of sequence-specific transcription factors, acting in a multifaceted manner. Upon PARylation, some factors, such as YY1, p53, CREB, Sp1, SRY and C/EBPβ, lose the ability to bind to their recognition sequences58. This function was initially underestimated, due to the absence of evident phenotypes, apart from the hypersensitivity to DNA damage, in PARP1 knock-out mice, probably as a consequence of redundant and/or compensatory roles of other PARP family members59. Further investigations, based on cellular models of differentiation, demonstrated that PARP1 regulates gene expression at least during adipogenesis60, osteoclastogenesis61 and neuronal differentiation49, as well as during induced pluripotency62. Despite the large amount of evidence that PARP1 modulates different epigenetic events, most of which also play critical roles in MyoD-dependent transcription, little, if any, is known about the roles of PARP1 in muscle cells, apart from the impact of PARP activity on the cellular bioenergetics of differentiated myotubes64.As expected for such a pleiotropic regulator of transcription, it is emerging that PARP1 plays an important role in some developmental and differentiation processesIn the present work we provide evidence that PARP1 participates in the transcriptional control of some paradigmatic examples of MyoD target genes by interfering, independently of its catalytic activity, with the recruitment of this myogenic factor to their regulatory regions within chromatin.63. According to this observation, and as shown in Supplementary Fig. p57 and myogenin, two examples of direct MyoD targets. This observation hinted at a possible involvement of PARP1 in the control of the muscle differentiation process. To explore this hypothesis, we first examined the effects of PARP1 depletion on muscle gene expression in C2.7 cells stably knocked-down for PARP1. For this purpose, we employed a short hairpin RNA for PARP1 (shPARP1) cloned into a retroviral vector as previously described45. Cell pools stably transduced with the shPARP1 or the empty control vector were isolated by puromycin selection and tested for the knockdown efficiency. As reported in Fig. p57 and myogenin expression in differentiated cells. A similar effect was observed on the induction of p21, another Cip/Kip inhibitor, and of Mef2C, another muscle-regulatory factor, both directly up-regulated by MyoD at early stages of muscle differentiation1. The observation that PARP1 depletion did not affect the basal, but only the induced expression of these genes, is easily explained by the well-established occurrence of multiple repressive mechanisms that prevent MyoD activity in proliferating myoblasts65 and suggests that PARP1 participates in keeping under control the MyoD-target genes during the differentiation process.It has been previously reported that PARP1 expression is down-regulated during muscle differentiation, allowing differentiated myotubes to acquire resistance to oxidative stress65, were not affected at all by PARP1 –positive myotubes, which is a later event of differentiation. This effect was observed both in depletion and in re-expression experiments . This feature allows the correct function of the human protein in mouse cells and, at the same time, reduces the efficiency by which the short hairpin targets the exogenous PARP1 RNA. shPARP1 myoblasts were co-transfected with either the puc19—human PARP1 vector or with the empty vector, together with a selection vector containing the hygromycin resistance gene. Stable cell pools were selected by hygromycin treatment and first checked for the re-establishment of PARP1 expression. As reported in Fig. nts Fig. . The impp57 distal regulatory region KVDMR1 and on myogenin promoter, critical and well-characterized elements for the differentiation-dependent expression of the two genes. As regards KvDMR1, we have previously demonstrated that this region, unlike p57 promoter, is directly bound by MyoD upon differentiation and that its epigenetic status influences the recruitment of the myogenic factor and the consequent disruption of a repressive chromatin loop with p57 promoter19. Concerning myogenin promoter, this region has always been a paradigmatic model for the study of MyoD-induced transcription and has been characterized in great detail for the dynamics of histone modifications and transcription factor binding during differentiation67. Nuclear extracts were collected from undifferentiated and differentiated cells and analysed for the enrichment of KvDMR1 and myogenin promoter in PARP1 immunoprecipitates. Rhodopsin (Rho) promoter was used as a negative control, as previously reported45. As shown in Fig. myogenin promoter, but not to Rho promoter, suggesting its involvement in the regulation of p57 and myogenin transcription. Moreover, in line with the decrease of the total protein levels observed during differentiation, the association of PARP1 with both regions was reduced in differentiated respect to undifferentiated cells, supporting the hypothesis that the binding of the enzyme to the regulatory regions of p57 and myogenin might inhibit their induction. A ChIP assay for MyoD binding to the same regions confirmed our previous findings and indicated the recicprocal occupancy of these two proteins. Moreover, as reported in Supplementary Fig. 19 and the second through direct MyoD binding15.To explore the molecular mechanism by which PARP1 represses gene expression in differentiating muscle cells, we performed a chromatin immunoprecipitation (ChIP) analysis of PARP1 interaction with chromatin during differentiation. We focused our attention on the myogenin promoter declines during differentiation, in concomitance with the onset of MyoD recruitment, we speculated that PARP1 could repress p57 and myogenin expression by interfering with the binding of MyoD to their regulatory regions. To verify this hypothesis, we evaluated the relationship between PARP1 and MyoD association with KvDMR1 and myogenin promoter in cells silenced for PARP1 and in cells re-expressing PARP1 compared to their respective controls. ChIP assays for the interaction of PARP1 or MyoD with KvDMR1 and myogenin promoter were performed in cells cultured in differentiation conditions, in which MyoD is normally recruited to these regions. As shown in Fig. As mentioned above, the impairment of MyoD recruitment to its binding elements, due to chromatin restraints, is involved in the failure of MyoD-dependent gene activation in undifferentiated cells or in cells refractory to express some specific MyoD target genes. Considering that the association of PARP1 with KvDMR1 and p57 and myogenin regulatory regions during differentiation. In light of the technical difficulties reported for the study of chromatin PARylation and, in particular, considering that cell lysis can induce DNA damage and artificial PARylation of non-specific chromatin targets 68, we also analysed two additional regulatory regions, as negative and positive controls respectively. One is Rho promoter, that we have previously shown to be scarcely PARylated 45. The other one is Igf2 DMR1 , which was previously reported to be susceptible to PARylation 69. As reported in Fig. myogenin promoter represented a first indication that PARylation did not play a role in their regulation.To investigate the possible role of the enzyme activity in the observed effects, we first measured the total PAR levels during differentiation. Unexpectedly, as demonstrated by the western blot analysis reported in Fig. p57 and myogenin by PARP1 was independent of the enzyme activity, we measured the expression of the two genes in myoblast cells induced to differentiate in the presence of pharmacological PARP inhibitors. To this end we used the small molecules ABT-888 (Veliparib) and AZD-2281 (Olaparib) two PARP inhibitors selective for PARP1 and PARP2 family members70 and capable of strongly inhibiting PARylation at concentrations that do not affect cell viability71. As expected, and as reported in Fig. p57 and myogenin induction , an active chromatin mark. PARP1 was reported to affect H3K4me3 in a complex manner. In particular, in its active state, the enzyme inhibits the H3K4me3 histone demethylase KDM5B, promoting the accumulation of this modification on multiple gene promotersIn the present work we provide evidence, for the first time, that PARP1 participates in the control of gene expression during muscle differentiation and, in particular, we show that PARP1, independently of its enzymatic activity, functions as negative regulator of MyoD binding to chromatin. These findings not only add new insight into the transcriptional control of muscle differentiation, but also expand our knowledge about the differentiation-related functions of PARP1.p57, myogenin, Mef2C and p21, while PARP1 re-expression reduces their up-regulation. The small, yet statistically significant changes in gene expression, observed when PARP1 levels are modulated, are accompanied by similarly small changes in later events of differentiation, such as the fusion into myotubes. However, it is conceivable that a refined modulation of muscle gene expression may play a critical role during in vivo processes of myogenesis, such as muscle development or regeneration, which take place over more extended time periods respect to in vitro differentiation.We found that PARP1 depletion promotes the induction of Importantly, our finding that PARP1 affects at least four different genes, which are known to be induced with different kinetics and through different molecular mechanisms, but which share the property to be directly targeted by MyoD, strongly supports a functional interference of PARP1 with MyoD-dependent transcription.75 and in human embryonic stem cells76 as well as for specific genes such as PARP1 itself in Ewing's sarcoma cells77 and IL6 in the RAW 264.7 macrophage cell line 74. Accordingly, we found that PARP1 directly interacts both with myogenin promoter, the prototypical regulatory region targeted by MyoD67, and with KvDMR1, a distant imprinting control region that we identified as a MyoD-responsive element involved in the control of p57 induction during differentiation19. Moreover, consistent with the decrease of total PARP1 levels and with the need of relieving a negative control from these genes to allow their up-regulation, PARP1 binding was significantly reduced in differentiated respect to undifferentiated cells. It is worth noticing that the loss of PARP1, although necessary, does not appear sufficient for inducing MyoD targets, as indicated by the observation that the basal expression of these genes, in undifferentiated cells, was not up-regulated upon PARP1 depletion. However, this is not surprising when considering that MyoD, although expressed since before differentiation, is kept inactive by interacting proteins and post-translational modifications induced by proliferation pathways65. For this reason, although it is possible that PARP1 participates in the repression of MyoD-targets in undifferentiated cells, the principal suggestion emerging from our data is that the role of PARP1 is to limit the up-regulation of muscle genes during differentiation  (Gibco) supplemented with 10% fetal bovine serum (FBS) (Microgem) and maintained sub-confluent in order to avoid differentiation. To induce MyoD activity and differentiation, cells were shifted to DMEM supplemented with 0.5% FBS (differentiation medium) and collected 24 or 48 h later, as indicated. 1 µM Olaparib (Selleckchem) or 1 µM Veliparib (Selleckchem) were administered to cell cultures at the time of the shift to differentiation medium.45.The shPARP1 retroviral vector was previously constructed, as described45. For the stable knock-down of PARP1, C2.7 cells were incubated with the BOSC23 retroviral supernatant supplemented with 4 μg/ml polybrene for 8 h and then re-fed with fresh medium. 48 h after infection cells where seeded at low density in selective medium containing 2.5 μg/ml of puromycin (Sigma) and, after about one week, the surviving clones were pooled together and cultured as indicated for C2.7 cells.Empty (Control) and shPARP-1-expressing retroviruses were produced by transfecting the constructs into BOSC 23 packaging cells as previously reportedStable re-expression of PARP1 was obtained by co-transfecting the puc19-PARP1 plasmid, coding for human PARP1 and kindly provided by Prof. Paola Caiafa, with the pSV2hygro plasmid, coding for the hygromycin resistance, in ratio 10:1, into shPARP1 C2.7 cells, using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. 48 h later, cells transfected with either puc19-PARP1 plasmid or with the empty vector puc19 were seeded in selective medium containing 2.5 μg/ml of hygromycin (Sigma) and, after two weeks, the surviving clones were pooled together and cultured as indicated for C2.7 cells.Cells were collected in RIPA buffer  or in Laemmli 1X buffer . Proteins were resolved by electrophoresis in 8% polyacrylamide SDS-PAGE and transferred to nitrocellulose membranes by electro-blotting. Membranes were blocked in 2% non-fat dry milk in TBS containing 0.05% Tween 20 for 1 h at room temperature and incubated with the primary antibodies overnight at 4 °C, then washed three times and incubated with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad). Proteins were detected using the ECL chemiluminescence system (Cyanagen).The following primary antibodies used for western blot were purchased from Santa Cruz Biotechnology: anti-PARP1 , anti-PAR , anti-p57  and anti-ERK1 . The F5D hybridoma supernatant was used for myogenin.Total RNA was extracted using the High Pure RNA Isolation Kit according to the manufacturer's instructions (Roche Diagnostics) and 500 ng were reverse transcribed with the iScript cDNA Synthesis Kit (Bio-Rad). Realtime-PCR (qPCR) reactions were performed in 10 μl of reaction buffer containing 1 μl of diluted cDNA in 9 μl of GoTaq qPCR Master Mix (Promega) containing each primer at the optimized final concentration (150-250 nM). The reaction was performed in the termocycler “MiniOpticon Real-Time PCR detection system” (Bio-Rad) or "CFX96 Real-Time PCR detection system" (Bio-Rad). The primer pair efficiency, the normalized expressions (ΔΔCt) and the Standard Error for the Normalized Expression were determined with CFX ManagerTM software (Bio-Rad).p57F: 5′-AACTTCCAGCAGGATGTGCC-3’R: 5′-CATCCACTGCAGACGACCAG-3′;for myogeninF: 5′-GTCTCTTCCTGAAGCCAGTTGCG-3′R: 5′-TGCAAATGCTTGGCCCCCAGAG-3′;for p21F: 5′-AGCAGCCGAGAGGTGTGAGC-3'R: 5′-TGGACATGGTGCCTGTGGCTCCT-3'for Mef2CF: 5′-TCCAAGTGCAGGTAACACAG-3'R: 5′-TTGCCGTATCCATTCCCTG-3'for Tbp, used as housekeeping geneF:5′-GGCGGTTTGGCTAGGTTT-3'R: 5′-GGGTTATCTTCACACACCATG-3’.for The following primer sets were used:19. After determining the DNA concentrations, each chromatin sample was divided into aliquots corresponding to 150, 75 and 50 μg of DNA.Nuclear lysates were prepared as previously describedChIP assays for PARP1, PAR and MyoD were carried out as previously described using 150 µg of chromatin for MyoD, and 75 µg for PARP1 and PAR. ChIP assays for H3K4me3 were performed using 50 µg of chromatin and Magna ChIP Protein A Magnetic Beads (Millipore) according to the manufacturer's instructions.The following antibodies were used for immunoprecipitation: anti-PARP1 ; anti-PAR , anti-MyoD , anti H3K4me3 , normal mouse IgG Antibody  and normal rabbit IgG Antibody .F: 5′-GCACAAGTCGCAAGTCCGCG-3’R: 5′-ATGGAGCCCAGCCGCGAAAG-3;for KvDMR1myogenin promoterF: 5′-TGGCAGGAACAAGCCTTTTGCGA-3’R: 5′-AGTCCGCTCATAGCCCGGGG-3′;for F: 5′-TTGTCAACTGGAGCCATGTG-3'R: 5′-GTGTCTCATCTTGTCTGCCC-3'for Rho promoterF: 5′-TGGAATGAGGAACATCACCA-3’R: 5′-TCTATCCCTGGCTTTTCTGG-3’for Igf2 DMR1qPCR analysis was performed in triplicate for each The following primer sets were used for qPCR analysis:For chart making and for statistical analysis, comparisons were performed using parametric paired Student's t-test analysis with a confidence interval of 95%, using GraphPad Prism® graphic and statistical software. Statistical significance is shown as p < 0.05 (*) or p < 0.01 (**) or p < 0.001 (***).Supplementary file1


Wap super-enhancer to activate the promoter of the juxtaposed and ubiquitously expressed Tbrg4 gene in the mouse mammary gland. The Wap super-enhancer was fused, alone or in combination with the Wap promoter, to the Tbrg4 gene. While the super-enhancer increased the expression of the Tbrg4 promoter five-fold, the combination of the super-enhancer and promoter resulted in 80-fold gene upregulation, demonstrating lineage-specific promoter–enhancer synergy. Employing ChIP-seq profiling to determine transcription factor binding and identify activating histone marks, we uncovered a chromatin platform that enables the high-level expression of the native promoter–enhancer but not the heterologous promoter. Taken together, our data reveal that lineage-specific enhancer–promoter synergy is critical for mammary gene regulation during pregnancy and lactation.Lineage-specific genetic programs rely on cell-restricted super-enhancers, which are platforms for high-density transcription factor occupation. It is not known whether super-enhancers synergize specifically with their native promoters or provide autonomous and independent regulatory platforms. Here, we investigated the ability of the mammary  Super-enhancers are regions of DNA that highly activate the expression of specific genes. Research led by H.K.L. and L.H. at the National Institutes of Health, USA, now demonstrate that a super-enhancer is much more effective when operating in synergy with its associated promoter, another gene-controlling element. They investigated super-enhancer and promoter synergy in mouse mammary gland cells. A mammary super-enhancer achieved a 5-fold activation of a gene with a common promoter, in contrast to an 80-fold activation with its own promoter together. The findings reveal that this synergy is critical for gene regulation in the mammary gland during pregnancy and lactation. They also help resolve the general issue of whether super-enhancers act independently or, as found here, can work together with a specific promoters in cell-specific genetic programs. They feature platforms with a high density of transcription factors (TFs) and other regulatory components, such as RNA polymerase II (Pol II). However, it remains unclear why tissue-specific genes associated with structurally equivalent SEs are expressed at widely different levels, ranging by several orders of magnitude. SEs have been extensively investigated in the mammary gland, where they have been linked to genes induced to high levels of expression by lactation hormones during pregnancy2. The cytokine-induced signal transducer and activator of transcription (STAT) 5 TF is at the core of mammary SEs, which anchors additional TFs, including the glucocorticoid receptor and nuclear factor IB (NFIB)4. RNA-seq experiments have revealed that the expression of genes associated with mammary SEs ranges from less than one Fragments Per Kilobase of transcripts per Million mapped reads (FPKM) to more than 500,000 FPKM, and more than one-half of these expressed genes are not induced during pregnancy2. We speculated that this enormous diversity of gene expression is not only inherent to the structure and activity of the enhancers but is also caused by the interplay of specific and unique promoter–enhancers. Moreover, the impact of possible posttranscriptional regulation needs to be considered when assessing steady-state mRNA levels as surrogates for gene expression levels.Super-enhancers (SEs) are key components in the control of lineage-specific transcription programsDrosophila5 and the presence of promoter-specific transcriptional cofactors6, decisive genetic studies to validate promoter–enhancer synergy in vivo have not been conducted in mammals. As the mammary Wap-SE increases Wap gene expression by 1000-fold during pregnancy, we have investigated its ability, by itself and with its associated promoter, to induce the juxtaposed Tbrg4 gene that is expressed in a wide range of cell types. Here, we employed CRISPR-Cas9 genome engineering in mice and placed the Tbrg4 gene under the control of Wap regulatory elements. Our study provides genetic evidence that selective promoter–enhancer synergy is critical for the exceptional mammary-specific gene upregulation during pregnancy and lactation. Since the abundance of mammary-specific Wap mRNA during lactation cannot be attributed exclusively to transcriptional regulation, we also explored the possibility that the highly conserved 3′ untranslated region (UTR) plays a role. Finally, we provide evidence that the cAMP response element (CRE)-binding protein (CREB) TF is an integral part of non-mammary promoters and modulates the Tbrg4 gene in vivo.Although enhancer–promoter specificity was suggested by self-transcribing active regulatory region sequencing (STARR-seq) of https://oacu.oir.nih.gov), and all animal experiments were approved by the ACUC of National Institute of Diabetes and Digestive and Kidney Diseases  and performed under the NIDDK animal protocol K089-LGP-17.Six- to eight-week-old C57BL/6 female mice were purchased from Charles River. CRISPR/Cas9-targeted mice were generated by the Transgenic Core of the National Heart Lung and Blood Institute (NHLBI). All animals were housed and handled according to the guidelines of the Animal Care and Use Committee (ACUC) of the NIH (http://crispr.mit.edu/). Sequences of the specific sgRNAs used are listed in Supplementary Table 2. The next morning, the embryos that had reached the two-cell stage were implanted into oviducts of pseudopregnant recipients. All the mice were genotyped after PCR amplification of genomic DNA isolated from the tip of the tail and Sanger sequencing. The PCR and sequencing primers used are listed in Supplementary Table CRISPR small-guide RNA (sgRNA) constructs were designed based on their proximity to the mutation sites and their off-target scores , day 1 of lactation (L1), and day 10 of lactation (L10) were ground into powder with a mortar and pestle. The chromatin was fixed with 1% formaldehyde at room temperature for 10 min, and the fixation was quenched with glycine at a final concentration of 125 mM. The samples were processed as previously described. The following antibodies were used for ChIP-seq: anti-STAT5 , anti-phospho-CREB , anti-H3K27ac , anti-H3K4me3 , and anti-RNA Polymerase II . Libraries for next-generation sequencing (NGS) were prepared as previously described and sequenced with HiSeq 2500 (Illumina).7 (version 0.36) and Bowtie8 (version 1.1.2), with the parameter m1 selected to retain only uniquely mapped reads, using the mm10 reference genome. Picard tools  were used to remove duplicates, and subsequently, Homer9 (version 4.8.2) software was applied to generate bedGraph files. The integrative Genomics Viewer10 (version 2.3.81) was used for visualization. Each ChIP-seq experiment was conducted with two replicates.Quality filtering and alignment of the raw reads was performed using trimmomatic9.The reads from Pol II ChIP-seq covering each gene were then counted and normalized to those of the library size and gene length, and they were used to rank the transcription levels of SE-associated genes. Then, a motif search was performed with the promoters of the top 50 most-transcribed genes with those of the 50 least-transcribed gene promoters as background in Homer. Pol II and H3K27ac signaling on the promoters associated with different motifs was also profiled in HomerTotal RNA from the L10 mammary glands of Wap-Tbrg4 heterozygous mice was isolated using a PureLink RNA Mini kit (Ambion). RNA quality was measured by a bioanalyzer (Bio-Rad). One microgram of total RNA from each sample was used to generate the sequencing library with a TruSeq Stranded Total RNA Library Prep kit (Illumina) according to the manufacturer’s protocol. The RNA-seq library was sequenced with a HiSeq 2500 system (Illumina).11. The UCSC known gene database was used as a gene coordinate reference. To count the RNA-seq reads on exons and introns, the exons from all the transcripts of each gene were collapsed. The intron coordinates for each gene were then calculated based on the collapsed exon coordinates. HTSeq was used to count the RNA-seq reads on the exons and introns, and FPKM values were generated using the DESeq2 package in R (http://www.R-project.org)12.RNA-seq data were mapped to the mm10 reference genome using the STAR alignerWap, Mm00839913_m1, Thermo Fisher Scientific; mouse Gapdh, 4352339E, Applied Biosystems) on a CFX384 Real-Time PCR detection system (Bio-Rad) according to the manufacturer’s instructions. Tbrg4_Wap-fused mRNA and Wap 3′UTR-inserted Tbrg4-3′UTR mRNA were measured with the following respective primers using SYBR Green system: forward, 5′-TTTCCCAGTAATCCCAGTGC-3′, and reverse, 5′-CAAGGAGAAACGCTGTCTAGG-3′, and forward, 5′-CTGCAGGCTTCCTGGTAGTAGAT-3′, and reverse, 5′-CTTTTCGCATCTTGTCCTTGAG-3′. The 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 completed in triplicate, and the data were normalized to those of the housekeeping gene Gapdh. Relative differences in PCR results were calculated using the comparative cycle threshold (CT) method.Mammary tissues were harvested at the p14, L1, and L10 time points and then processed by an electronic homogenizer. Then, RNA was extracted using a PureLink RNA Mini kit (Ambion) according to the manufacturer’s instructions. cDNA was synthesized from total RNA using Superscript II (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using TaqMan probes  is ubiquitously expressed at low levels13. Here, we investigated whether the Wap-SE has an inherent preference for its associated promoter or has the capacity to effectively activate unrelated promoters. Tbrg4 and Wap are positioned in the same orientation and are separated by 13 kb. We used CRISPR-Cas9 to delete the sequences between the Tbrg4 and Wap promoters in the mouse genome, thereby fusing Wap regulatory elements with the downstream Tbrg4 gene , was translocated to the body of Tbrg4, thus generating a hybrid gene. An analysis of lactating mammary tissue from the SE-Tbrg4 mice revealed a five-fold increase in Tbrg4 mRNA  (unpublished data), suggesting that Tbrg4 expression in non-mammary cells is essential for embryonic development. To permit further analysis of the mutant allele, we generated compound heterozygous mice carrying one copy of the intact Tbrg4 gene with a silent Wap gene and one copy of the Wap-Tbrg4 fusion gene  is no longer required, and the two distal enhancers (E2 and E3) are sufficient for Wap gene expression3. To evaluate whether the two distal enhancers have the capacity to activate the Tbrg4 promoter, we deleted ~18 kb of DNA separating the Tbrg4 promoter from its upstream region and the central constituent enhancer (E2) from Wap-SE  and day 1 of lactation (L1) and another 5-fold at L10. Wap mRNA levels from the WT allele increased ~5000-fold between p14 and L1 and another 10-fold at L10  . However, the levels of Pol II loading and Glycam1 transcription were low (average nascent RNA FPKM 0.5)  or sub-TAD29. In a previous study13, we identified two CTCF sites that coincide with a TAD boundary30 and separate the Wap gene from the Tbrg4 gene. Deletion of the two CTCF sites did not result in altered expression of either Tbrg4 or Wap in lactating mammary tissue13, demonstrating that CTCF at either site does not insulate the Tbrg4 gene from Wap-SE. Since no additional potential insulating elements were identified between Wap-SE and Tbrg4, we speculate that distance might play a larger role than previously thought. Alternatively, Wap-SE might preferentially recognize the most proximal promoter.Although expressed in mammary tissue, the Our findings that enhancer-promoter synergy determines transcription output provide an explanation for the distinct transcriptional levels of SE-associated genes. The expression of the more than 400 genes that are under the control of mammary SEs varies over several orders of magnitude, suggesting the presence of additional promoter-bound TFs that can greatly boost mammary SE activity. We identified CREB TF binding in non-mammary promoters but not in promoters selectively active in mammary tissue, suggesting that they might suppress the promoter response to mammary SEs. However, genetic ablation of CREB binding on a non-mammary promoter did not support this concept.32. Similar to milk mRNAs stabilized by hormones in mammary tissue, vitellogenin mRNA is stabilized by estrogen in the liver, while interferon and IL-2 are stabilized by CD3 and CD28 receptor involvement in T cells34. However, the mechanisms of hormone-mediated RNA stabilization are still unclear. Hormones may trigger specific RNA-binding proteins to protect mRNA from degradation or activate/inhibit specific small RNAs that regulate RNA decay35. Lineage-specific and gene-specific mRNA stabilization probably involves both of these processes and requires further investigation.Differential mRNA half-lives were discovered decades agoWap gene, including its promoter and SE, it also shed light on the Tbrg4 gene and its role in mammalian development. TBRG4, also known as FASTKD4, is transported to mitochondria and serves as a key posttranscriptional regulator of mitochondrial gene expression36. Notably, it appears to control the half-lives of mitochondrial RNAs38. In cell lines, CRISPR-mediated targeting of the Fastkd4 locus resulted in an apparent absence of the corresponding protein and decreased levels of mitochondrial RNA, but no aberrant cell viability was reported36. Tbrg4 is expressed across all cell types, and in our study, the Tbrg4 allele was expressed only in the mouse mammary epithelium. While the heterozygous mice were viable and indistinguishable from the WT mice, homozygous mutant mice were not viable, with the fetuses dying prior to implantation, demonstrating that Tbrg4 function is essential for early embryonic development. It is speculated that impaired mitochondrial physiology causes the early embryonic death. Our mouse study highlights differences in the requirements of this mitochondrial regulator in the fast-growing embryo compared with those in the immortalized cell lines that survive without TBRG439.While our study focused on understanding the mechanistic aspects of the Supplementary informationSupplementary Table 5


Historically, the mammalian brain was thought to lack stem cells as no new neurons were found to be made in adulthood. That dogma changed ∼25 years ago with the identification of neural stem cells (NSCs) in the adult rodent forebrain. However, unlike rapidly self-renewing mature tissues , the majority of adult NSCs are quiescent, and those that become ‘activated’ are restricted to a few neurogenic zones that repopulate specific brain regions. Conversely, embryonic NSCs are actively proliferating and neurogenic. Investigations into the molecular control of the quiescence-to-proliferation-to-differentiation continuum in the embryonic and adult brain have identified proneural genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs) as critical regulators. These bHLH TFs initiate genetic programs that remove NSCs from quiescence and drive daughter neural progenitor cells (NPCs) to differentiate into specific neural cell subtypes, thereby contributing to the enormous cellular diversity of the adult brain. However, new insights have revealed that proneural gene activities are context-dependent and tightly regulated. Here we review how proneural bHLH TFs are regulated, with a focus on the murine cerebral cortex, drawing parallels where appropriate to other organisms and neural tissues. We discuss upstream regulatory events, post-translational modifications , protein–protein interactions, epigenetic and metabolic mechanisms that govern bHLH TF expression, stability, localization, and consequent transactivation of downstream target genes. These tight regulatory controls help to explain paradoxical findings of changes to bHLH activity in different cellular contexts. One-hundred years ago, Santiago Ramon y Cajal likened the pyramidal cells of the cerebral cortex to “a garden filled with innumerable trees…which can multiply their branches thanks to intelligent cultivation, send their roots deeper, and produce more exquisite flowers and fruits every day” . In his This review centers on the regulatory events that govern proneural gene function in the developing and adult cerebral cortex. To provide a contextual framework, we first provide a high-level overview, not meant to be comprehensive, of the cellular context in which these genes function. The cerebral cortex, which is the seat of higher order cognitive functioning and sensory processing, is comprised of a six-layered neocortex and several three- or four-layered allocortical territories, including the hippocampal formation and paleocortex. Cortical territories are found in all mammals, but display enormous structural diversity across species, transitioning during evolution between smooth  structures in smaller mammals such as rodents, to highly folded  structures in most extant primates and larger mammals . These gTo understand how the cerebral cortex acquires species-specific forms, it is essential to elucidate how NSC/NPC (hereafter NPC for simplicity) fate decisions are controlled. Proneural genes, which encode basic-helix-loop-helix (bHLH) transcription factors (TFs), are critical pieces to the puzzle as they control NPC decisions to divide or differentiate while also specifying neural subtype identities . At faceDrosophila melanogaster where they belong to two main families that each specify distinct neural cell fates: achaete-scute complex (AS-C) and atonal-related genes (Dll1 and Dll3 (Neurogenin (Neurog) 1, Neurog2, Neurod4 (aka Math3), and Achaete-scute family bHLH transcription factor 1   . All oth Neurod1 ], and/or Neurod6 ]. In thiTcf4 (aka E2-2), Tcf12 (aka HEB), and Tcf3 (aka E2A), the latter encoding E12 and E47 splice variants  that make up the adult cerebral cortex are derived from multipotent NPCs located in the dorsal telencephalon (cortex), with some additional contributions from ventral telencephalic  NPCs. Telencephalic NPCs are parcellated into apical and basal compartments . Apical Cortical NPCs give rise in a sequential fashion to excitatory glutamatergic neurons that form the six layers of the cortical plate between E11-E17 in mouse , followeearly cortical NPCs are multipotent and give rise to neuronal clones that span cortical layers, whereas late NPCs are fate restricted and only generate upper layer neurons  gene as it is expressed at high levels in apical and basal NPCs and at low levels in newborn neurons  cortical neurons, as well as Cajal-Retzius neurons, which populate layer I  and human cortices have revealed that the apical NPC pool has expanded to include both aRG and outer or basal RG (bRG), the latter forming a large outer SVZ (oSVZ) not present in rodents . Like aRectories .While NSCs persist into adulthood, they differ from embryonic NSCs in several ways. Firstly, the adult NSC transcriptional profile is more closely related to astrocytes than to embryonic NSCs . Secondlin vivo, with cell cycle times of 8–18 h (+ periglomerular cells (PGCs) and granule cells (GCs), and septal NSCs to calretinin+ PGCs and GCs  1 and Hes5, which encode bHLH transcriptional repressors that recruit Groucho/TLE co-repressors and bind to N-boxes (CACNAG), directly repressing proneural gene transcription to form a lateral inhibitory loop , as revealed by the mutation of several cortical transcription factors , or whenemselves , they are cortex , while ccal NPCs . Neurog2or cells . Given tof Ascl1 . Notablynd Ascl1 , as Neur Neurog1 . Howeveregulated , indicatNeurog2 and Ascl1 are also cross-repressive at the functional level; Neurog2 inhibits the ability of Ascl1 to promote a glioblast fate, while Ascl1 inhibits the ability of Neurog2 to specify a glutamatergic neuronal identity in cortical NPCs  (nerve growth factor (Ngf) (neurotrophin 3 (Ntf3) (brain derived neurotrophic factor (Bdnf)  , nerve gor (Ngf) , neurotr3 (Ntf3) , and brar (Bdnf) . Each ofr (Bdnf) . Mechanir (Bdnf) , where Fr (Bdnf) . Mechanir (Bdnf) .FGFR3) and a set of ets-domain transcription factors activated downstream of RTK signaling, including Etv1, Etv4 and Etv5  (Thr202/Tyr204), which is initially detected in the antihem adjacent to the lateral pallium where neurogenesis is first initiated in the cortex , before and Etv5 . The Etved above . Taken tCtnnb1) is stabilized and translocates to the nucleus where it forms active transcriptional complexes with Tcf1. Conditional knock-out (cKO) of Ctnnb1 in early cortical NPCs, prior to neurogenesis, downregulates Neurog2 and upregulates Ascl1 expression  signaling in specifying a cortical identity, two transgenic reporters for this pathway, BAT-gal  and TCF-pression . Converspression , to mainin vitro . Similares Ascl1 . Thus, tell fate .Neurog2 function is attenuated , correlating with the time when tenuated . In the tenuated , which ptenuated . Notablytenuated , but ratSeveral signaling pathways induce cortical NPCs to differentiate into astrocytes , includiNeurog1/2 and/or Ascl1 are biased against an astrocytic fate  and precocious neurogenesis that occurs in E12.5 Neurog1–/– cortices  as well as the ability of Neurog2 to induce in vivo glutamatergic neuron formation  or Ascl1 (DeltaM)  . Alternapromoter . FurtherRnd2, a critical regulator of cortical neuron migration  promotes the formation of TF complexes between Neurog2 and the adaptor protein Ldb1, which recruits LIM-homeodomain TFs Isl1 and Lhx3 to form a complex that transactivates motor neuron specific genes . Similarectively   junction acts as an evolutionarily conserved, binary ‘off’ switch for both vertebrate and invertebrate proneural TFs  Figure . 3D modein vivo . Strikinin vivo , b. The in vivo .Neurog2 is also phosphorylated on Tyr241, a residue outside the bHLH domain that is dispensable for proneural activity, but required to specify a polarized neuronal phenotype and establish appropriate radial migration patterns . Mutatioscute (Sc); the transactivation domain (TAD) and an SPTSS motif, including a phosphoacceptor site for proline-directed S/T kinases  . UbiquitNSC pool . SpatialNSC pool . CytoplaNSC pool .While comparable E3-ligases have yet to be identified for Neurog1 and Neurog2 in the cortex, Fbxo9 is an E3-ligase that destabilizes Neurog2 in the developing dorsal root ganglia . MechaniNeurog2 are temporally restricted; in the embryonic cortex, Neurog2 only efficiently induces neurogenesis before E14.5  chromatin and facilitate the opening of these sites for TF binding  Figure . leadingAscl1 was overexpressed in mouse embryonic fibroblasts (MEFs), Ascl1 induced CpH methylation of fibroblast-specific genes, but cooperation with other TFs   that are prevalent throughout the genome, including in non-regulatory regions, but these modifications similarly repress gene expression . Strikin, Myt1l) , which i, Myt1l)   .trans-differentiation of MEFs to neurons showed that Ascl1 induces widespread chromatin remodeling , as exemplified by p300/CBP . p300/CBOpening of the chromatin to provide access to TF binding is also associated with methylation of H3 histone tails, but here the specific lysine (K) residues are critical. For instance, while RNAPol II and trimethylation (me3) of histone H3K4 cluster at transcription start sites of actively transcribed genes, and histone H3K36me3 is in the body of actively transcribed genes, other chromatin marks are found in silenced regions of the genome  . NotablyOnly a handful of epigenetic gatekeepers are known; most influence chromatin structure, some via interactions with Polycomb group (PcG) proteins, which close chromatin . PcG proNeurog1, but not Neurog2, expression, with mutation of Ring1b extending Neurog1 expression into late-stage neurogenesis   . Extendical NPCs . Recentlcephalon . Surprispression . Ring1b pression , which it, Foxp2 . In thisEed (inactivation of PRC2) leading to precocious neurogenesis   . Similar period) .Overall, these findings implicate PcG proteins as crucial temporal regulators of proneural transcriptional programs, operating to limit the timeframe of subtype specification by proneural TFs by occluding proneural TF target genes. These critical epigenetic regulators do not act alone, though; temporal NPC fate specification is simultaneously and robustly regulated by other mechanisms such as declining proneural transcriptional activity.in vitro, as well as in vivo in injured mouse cortex  NPCs, proneural TFs may then be translated into TFs which are regulated in their stability and transcriptional activity by different dimerization partners and by phosphorylation of different residues within their bHLH, N- and C-terminal domains. When the actions of a proneural TF are no longer needed, the protein may be degraded proteolytically by polyubiquitylation (for which exact enzymatic candidates are emerging), or by broader inhibition at the epigenetic level .In the future, the ultimate application of the multifaceted picture of proneural gene regulation that we have painted in this review would aid in the refinement and improvement of neuronal reprogramming strategies incorporating these proneural genes for regenerative medicine purposes.A-MO and CS: conceptualization. A-MO, SH, and CS: writing—review and editing. A-MO and SH: artwork. CS: funding acquisition. All authors contributed to the article and approved the submitted version.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.


The role of genomic variation in differentiation is currently not well understood. Here, the genomic variations were determined with the whole-genome sequencing for three pairs of pluripotent stem cell lines and their corresponding BMP4-induced trophoblast cell lines. We identified ∼3,500 single nucleotide variations and ∼4,500 indels by comparing the genome sequenced data between the stem cell lines and the matched BMP4-induced trophoblast cell lines and annotated them by integrating the epigenomic and transcriptomic datasets. Relatively, introns enrich more variations. We found ∼45% (42 genes) of the differentially expressed genes in trophoblasts that associate genomic variations. Six variations, located at transcription factor binding sites where H3K4me3 and H3K27ac are enriched in both H1 and H1_BMP4, were identified. The epigenetic status around the genomic variations in H1 was similar to that in H1_BMP4. This means that the variation-associated gene’s expression change can not be attributed to epigenetic alteration. The genes associated with the six variations were upregulated in differentiation. We inferred that during the differentiation, an increased in the expression level of the MEF2C gene is due to a genomic variation in chromosomes 5: 88179358 A > G, which is at a binding site of TFs KLF16, NR2C2, and ZNF740 to MEF2C. Allele G shows a higher affinity to the TFs in the induced cells. The increased expression of MEF2C leads to an increased expression of TF MEF2C’s target genes, subsequently affecting the differentiation. Although genomic variation should not be a dominant factor in differentiation, we believe that genomic variation could indeed play a role in the differentiation from stem cells into trophoblast. Stem cell differentiation involves a complex but poorly understood biological process. Genetic and epigenetic factors have in the past been intensively studied for this process. Some key transcription factors (TFs) affecting differentiation have been identified, such as POU5F1, SOX2, and KLF4 . Previouin vivo. Since Xu et al. proved that bone morphogenetic protein 4 (BMP4) could induce human ESCs, efficiently differentiating to trophoblast lineage, multiple research institutes have adopted this system as a model to study trophoblast lineage specification in vitro  . We demoGenomic DNA sequences were determined for three stem cells and their corresponding BMP4-induced cell lines , in whic® spectrophotometer  (OD260/OD280). The Optical Density (OD) value of the qualified sample ranged between 1.8 – 2.0. (2) DNA degradation, and suspected RNA/Protein contamination were verified by electrophoresis on 1% agarose gels. (3) The concentration and purity of DNA samples were further quantified precisely by the Qubit DNA Assay Kit in Qubit®2.0 Flurometer . A total amount of 1 μg DNA per sample was required for library generation.The quality of isolated genomic DNA was verified using these three methods: (1) DNA purity and concentration were identified by NanoPhotometerPaired-end DNA libraries were prepared according to the manufacturer’s instructions (Illumina Truseq Library Construction). First, 1.0 μg Genomic DNA was sheared into an average size of 350 base pair (bp) fragments by Covaris S220 sonicator. Second, the ends of the gDNA fragments were repaired; 3′ ends were adenylated. Both ends of the gDNA fragments were ligated at the 3′ ends with paired-end adaptors (Illumina) with a single ‘T’ base overhang, and purified using AMPure SPRI beads from Agencourt. The size distribution and concentration of the libraries were then determined using Agilent 2100 Bioanalyzer and were qualified by real-time PCR (2 nM), respectively. Lastly, DNA libraries were sequenced on Illumina Hiseq X according to the manufacturer’s instructions for paired-end 150 bp reads.The raw image files obtained from the Hi-Seq platform were processed with the Illumina pipeline for base calling and stored as FASTQ format (Raw data). Quality control (QC) was as follows: (1) to filter reads with adapter contamination . (2) to discard the reads containing more than 10% uncertain nucleotides. (3) to discard the paired reads when a single read has more than 50% low quality nucleotides .1 with default settings to sort reads, remove duplicated reads, and to generate the final bam file. If one or more pair read(s) had multiple mapping positions, the best one was selected. If there were multiple best mapping positions, we randomly picked one.After quality control, the sequenced reads were mapped to the GRCh37 assembly of the human genome by Burrows-Wheeler Aligner (BWA) software using default settings . SubsequThe analysis flowchart is shown in Genomic variations were identified by comparing genomic DNA sequences between stem cell lines and matched BMP4-induced trophoblast cell lines using the following steps. Single nucleotide variation (SNV) and small somatic insertions and deletions (indels) were identified using the Strelka2  tool. AlThe ANNOVAR tool was used to produce statistical analyses of the SNVs/indels . The varThe regions include exon, intron, intergenic region, UTR5, UTR3, and the upstream (variant overlaps 1-kb region upstream of transcription start site)/downstream (variant overlaps 1-kb region downstream of transcription end site) regions.The annotation detail of these three cell lines is listed in 2 and retrieved from the GEO database according to their accession number  data were mapped to GRCh38/hg38  Roadmap. We usedp-value < 0.05 were identified as significantly differentially expressed genes between ESC H1 and differentiated to trophoblast H1_BMP4.The gene expression data  were retTo estimate the change of the TF binding affinity due to the genomic variation, the 150-bp DNA sequences were extracted around the genomic variations and inputted into a bioinformatics tool, HOMER , to calcWe performed the high-throughput DNA sequencing for three pluripotent stem cell lines  and differentiated trophoblasts . After quality control , genomicThe frequency of SNVs was counted. According to our statistics, the single-base substitution patterns were similar in the three pluripotent stem cells . The higWe then annotated SNV and indels with ANNOVAR . The SNVDistribution profiles of SNV/indels in the 2500-bp genomic regions around the TSSs seemly occurred in a periodic pattern in the cell lines . SNP freA genomic variation may directly alter the affinity of TF’s binding as this variation occurs exactly at TFBSs, thus influencing the transcription levels of the downstream target genes of the TFs. We assessed the effect of the variations on the alteration of TF binding with HOMER . The 150A TF with its motif in one genomic region does not mean the TF indeed binds at the region in a cell because the TF maybe is not expressed in the cell. We therefore need to check if the TF is abundant enough in the cell, namely, to check if the expression level of the gene encoding the TF is high enough. To do this, a necessary step is to exclude those TFs with a low abundance. The expression of TF-encoding genes was shown for H1 and H1_BMP4 . Here, tWe identified the TFs that differ in affinity around the genomic variation sites between H1 and H1_BMP4. The TFs were chosen under conditions of TPM ≥ 3, motif score ≥ 10, and motif score count difference (abs) ≥ 10. We found three TFs , whose affinities near the genomic variations differ between H1 and H1_BMP4 cell lines. The three kinds of TFs have a higher motif score count (count of motif score ≥ 10) in H1 than in H1_BMP4. Interestingly, these TFs are widely known to be involved in trophoblast differentiation. For example, Zfp281 (Krüppel-like zinc finger transcription factor), a zinc finger transcription factor, which shapes the transcriptome of trophoblast cells and regulates early placental development, has also been investigated in a recent article . MoreoveIn short, we found that there were indeed some genomic variations at TFBSs.Genome variation was reported to be correlated to epigenetic alteration. Li et al. demonstrated that about 2/3 of eQTLs were due to variations that altered chromatin accessibility or histone marking . We studIn this analysis, the genomic variations were divided into four categories according to whether the variation site was at the peaks of the histone modifications and DNA methylation in H1 and H1_BMP4 . We counThe enriching regions (peaks) of DNA methylation and H3K27me3 were considered as “inhibited” regions. Modified regions (peaks) by H3K4me3 and H3K27ac indicate “promoter activated” and “enhancer inactivated” regions, respectively. By comparing the epigenomic states of the regions around the genomic variations between the stem cells and matched BMP4-induced trophoblast cell lines, 66 variations that do not associate an epigenomic alteration were identified . Next, wWe are interested in the gene expression change that is only caused by the genomic variation instead of the epigenetic variation. We therefore only focused on the genomic variations which are located in those genomic regions where epigenetic markers remain comparable between H1 and H1_BMP4.We identified 5,688 differentially expressed genes between H1 and H1_BMP4  . In the p-value ≤ 1E–5 and fold change ≥ 4 or ≤1/4). We found a total of 26 genes with variations in the differentiation process out of the 129 differential expression genes . There are 72 up-regulated (30.64%) and 22 down-regulated genes (9.36%). In the 94 BMP4-induced genes, 42 (44.68%) contain the genomic variations  in differentiation and placenta development. TNFAIP8 plays a role in immune homeostasis, inflammatory responses, tumor genesis, and development. TNFAIP8 is also highly expressed in most normal human tissues especially for immunity-related tissues like the placenta . INHBA e3 to calculate the motif score of 12-bp DNA sequences around the six SNVs/indels to the TFs that bind at those variation-harbored regions   .p-value ≤ 0.05, Functional annotations by Enrichr  on KEGG Briefly, the gene expression of MEF2C was significantly increased due to the high-affinity binding sites of TFs like KLF16, NR2C2, and ZNF740, a variation site located in MEF2C (chromosomes 5: 88179358 A > G). Further, the expression levels of these three TFs were not significantly different between H1 and H1_BMP4, suggesting that MEF2C’s expression increase is not caused by a rise in the abundance of the TFs of MEF2C, but due to the genomic variation . InteresNR2C2 is expressed in blastocysts and embryonic stem cells and can act as transcriptional activators in hESC . AltogetUnderstanding the genetic underpinnings of complex traits remains a major challenge in human genetics. In this study, we obtained paired genomic DNA sequences of human ESC and the trophoblast from three cell lines  through whole genome sequencing, and integrated the epigenomic  and transcriptomic datasets to investigate the impact of the genome variations in human ESC differentiation to the trophoblast. We found that the SNVs and indels generally tend to be located in the intron regions rather than in the other regions.We focused on the gene expression variation caused by genomic variation rather than the epigenetic variation. Six genomic variations were identified. One of them, located in MEF2C (chromosomes 5: 88179358 A > G), is a TF. This SNV increased the TF binding strength to regulate gene expression directly. Thereby, leading to an increase in the expression of downstream target genes affecting the differentiation of human ESC into the trophoblast. It suggested that the variations in the non-coding region played an important role in the differentiation process.The inducer, BMP4, is the most significant factor to differentiate to the trophoblast. BMP4 is able to inhibit the Activin/Nodal signaling pathway and activate the BMP signaling pathway, which is required for human ESCs to differentiate into trophoblasts . We showCRA002190, which is publicly accessible at https://bigd.big.ac. cn/gsa.The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive  at the BAll authors contributed to the study conception and design, analyzed and discussed the results. XS conceived the project and completed the core program. HaL, HoL, and YL performed the computational analysis. HaL and HoL wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evi1) was discovered in 1988 as a common site of ecotropic viral integration resulting in myeloid malignancies in mice. EVI1 is an oncogenic zinc-finger transcription factor whose overexpression contributes to disease progression and an aggressive phenotype, correlating with poor clinical outcome in myeloid malignancies. Despite progress in understanding the biology of EVI1 dysregulation, significant improvements in therapeutic outcome remain elusive. Here, we highlight advances in understanding EVI1 biology and discuss how this new knowledge informs development of novel therapeutic interventions. EVI1 is overexpression is correlated with poor outcome in some epithelial cancers. However, the focus of this review is the genetic lesions, biology, and current therapeutics of myeloid malignancies overexpressing EVI1.Ecotropic viral integration site 1 ( Evi1 was discovered by Mucenski et al. as a common site of ecotropic viral integration in mice that caused virally induced myeloid malignancies1. Through its rearrangements in human acute myeloid leukemia (AML), the human EVI1 gene was mapped to the long arm of chromosome 3 at q 26.2 (3q26.2)2. The EVI1 gene in humans is ~92% homologous to the mouse Evi12. EVI1 is encoded from the MDS1 and ecotropic viral integration site 1 (EVI1) complex locus (MECOM), which includes several alternative transcripts3. EVI1 exists either as a shorter single gene or as spliced to the short myelodysplastic syndrome 1 (MDS1) gene, present more than 350 kb upstream to EVI1, creating the longer MDS1-EVI1 gene3. The shorter isoform of EVI1 is abundant and oncogenic5. A truncated variant of the EVI1 transcript conserved in both mice and humans, EVI1∆324, lacks part of the first zinc finger domain and the ability to transform 8. EVI1 also contains an acidic domain at its C terminus5  protein that contains 1051 amino acids. EVI1 localizes to the nucleus and binds DNA through its zinc finger (ZF) domainsEvi1 transcript by mutagenesis in mice led to severe developmental defects in the heart and central nervous system, and homozygous mutants died at approximately embryonic day 10.59. Additionally, adult mice with conditional knockout of Evi1 had a marked reduction in their long-term hematopoietic stem cells (LT-HSCs), and upon transfer into irradiated mice were unable to engraft and repopulate efficiently10. The self-renewal ability of LT-HSCs is linked to EVI1 expression, and many LT-HSC-associated genes have EVI1 binding sites in their regulatory regions11. Furthermore, increased EVI1 expression is a common immortalizing factor of murine primary bone marrow after retroviral infection12. For example, MSCV integration promoted increased expression of EVI1 causing immortalization of immature myeloid cells, but they were unable to induce leukemia in transplanted hosts12. Thus EVI1 supports HSC self-renewal, but EVI1 expression alone is not enough to drive leukemogenesis13.Disruption of the full-length 15. EVI1 expression in primary mouse myeloid progenitor cells upregulated HSC-associated genes and decreased DNA replication and repair genes14. EVI1 transcripts are decreased in human CD34+ cells after stimulation of differentiation induced by cytokine administration, suggesting that downregulation of EVI1 is an important step in terminal differentiation of many hematopoietic lineages15. Forced expression of Evi1 in the mouse bone marrow cell line 32Dcl3 inhibits differentiation response to granulocytes and erythrocytes due to granulocyte colony-stimulating factor (G-CSF) and erythropoietin, respectively17. Extrinsic EVI1 expression blocked G-CSF-induced differentiation through transcriptional repression of the lineage-specific gene myeloperoxidase and the myeloid transcription factors C/EBPα  and RUNX1 18. Erythroid differentiation was blocked by EVI1 through binding and subsequent inhibition of transcriptional activity of the myeloid transcription factors GATA1 (GATA binding protein 1) and PU.1 (transcription factor PU.1)19. In the megakaryocyte lineage, EVI1 is expressed in early precursor cells20. In a transgenic mouse model recapitulating human inv3(q21q26) AML that overexpresses EVI1 and also has GATA2 haploinsufficiency, EVI1 and GATA2 dysregulation together skewed hematopoiesis toward the megakaryocyte lineage more so than EVI1 overexpression alone21. This suggests that EVI1 may work in concert with other factors to promote the megakaryocyte lineage21. In general, for most myeloid lineages, EVI1 functions to promote a stem or early progenitor transcriptional program14. Forced EVI1 expression maintains the stem-like program while simultaneously suppressing myeloid transcription factors involved in myeloid differentiation21. Notably, endogenous EVI1 is generally downregulated under normal differentiation15. However, the degree to which endogenous EVI1 blocks differentiation and what factors normally downregulate EVI1 during differentiation largely remain an unknown.In addition to LT-HSC self-renewal, expression of EVI1 blocks hematopoietic differentiation of the granulocyte, erythroid, dendritic, and monocytic lineagesMDS1, which formed a fusion protein with RUNX1 and/or EVI1 in several myelodysplastic syndrome (MDS) patients22. Currently, the function of the MDS1 protein itself is still unknown. In MDS1-EVI1, exon 2 of MDS1 is fused in-frame to EVI1 exon 2, which adds 188 amino acids upstream of the normal start codon of EVI1 in exon 322. A part of these extra N-terminal amino acids contains the PR domain, which shares homology with the B cell factor positive regulatory domain 1-binding factor (PRD1-BF1) and retinoblastoma binding protein RIZ112. The PR domain is related to a subset of the methyltransferase SET domains23  at the EVI1 promoter. In contrast cell lines with low EVI1 expression have enrichment of the repressive histone mark H3K27me3 (histone 3 lysine 27 tri-methylation)25.The region 5′ of EVI1 was localized to a 318 nucleotide region 5′ of the EVI1 transcription start site that does not contain a traditional TATA or CAAT box26 , RELA , GATA1, and MYB . Knockdown of RUNX1 and/or ELK1 in HEL cells decreased EVI1 mRNA and protein levels26. Furthermore, interactions between RUNX1 and EVI1 at the minimal promoter appear to positively regulate EVI1 activity26. MDS1-EVI1 and EVI1Δ324 bind further downstream of the minimal promoter of EVI1 and reduce its transcription27. MDS1-EVI1 and EVI1∆324 are reported to be co-expressed with EVI124. Although not further studied, present upstream of the minimal EVI1 promoter are the consensus binding motifs for GATA1, GATA2, and C/EBPα, suggesting that MDS1-EVI1 and EVI1∆324 may work in concert with other transcription factors to repress EVI127. CML (chronic myeloid leukemia) blast crisis patient-derived cells express high EVI1 and β-catenin levels28. Knockdown of β-catenin or its related co-transcription factor LEF1 (lymphoid enhancer factor 1) decreased EVI1 levels28. Bioinformatic analysis indicated two potential tandem LEF1/β-catenin-binding sites present 1.44 kb upstream of EVI1, which are bound by LEF1, as determined by ChIP assays28. Additional studies are needed to further clarify regulation of EVI1 by LEF1/β-catenin, RUNX1, GATA1, and/or ELK1.The minimal promoter of 30. Stable isotope labeling of amino acids followed by mass spectrometry (SILAC-MS) identified EVI1-associated proteins. CK2 (casein kinase 2) was confirmed to phosphorylate EVI1 residues S538 and S858. Loss of phosphorylation was mediated by PP1α , and it decreased DNA-binding by the C-terminal ZF domain29. In contrast, phosphorylation of S196 on the 6th zinc finger in the N-terminal ZF domain decreases DNA binding and repression by EVI1 of promoters containing GATA-like motifs30. Although phosphorylation of Ser858 and Ser860 did not affect EVI1 DNA binding, loss of these phosphorylations blunted EVI1 transcriptional repression after cellular stress through reduced interaction of EVI1 with co-repressor CtBP131. EVI1 is also acetylated by CBP (CREB binding protein or KAT3A)/p300  and PCAF (P300/CBP-associated factor or KAT2B)32. CBP-induced acetylation increased EVI1 transcriptional activity in luciferase assays32. In contrast, PCAF-mediated acetylation of EVI1 has been reported to exhibit opposing effects on EVI1 activity. Co-expression of EVI1 with PCAF abrogated EVI1-mediated Bcl-xL expression, suggesting that EVI1 acetylation blocked EVI1 transactivation activity at the Bcl-xL promoter33. In contrast, PCAF-mediated acetylation of K564 on EVI1 increased its ability to transactivate GATA2, and this ability was lost in a K564A mutant that cannot be acetylated34. Overall, it is unclear whether these post-translational modifications can occur simultaneously, or whether one modification can hinder the acquisition of another.EVI1 has been reported to be phosphorylated at serine 196 (S196), S538, S858, and S86036 (Table 36.EVI1 co-immunoprecipitates with the H3K9me3 methyltransferase SUV39H1 (suppressor of variegation 3–9 homolog 1) and the related H3K9me1/2 methyltransferase G9a (euchromatic histone lysine methyltransferase 2)36 Table . EVI1 an37. This increased accumulation of the repressive H3K27me3 mark and reduced histone acetylation at the PTEN locus has been observed in human AML patient samples37.EVI1 represses PTEN (phosphatase and tensin homolog) through its N-terminal ZF domain and via recruitment of the polycomb repressor complex 2 (PRC2), including EZH2 (enhancer of zeste 2), by binding upstream of the PTEN transcription start site39. EVI1 expression correlated with differential hypermethylation of over 200 genes, as compared to normal CD34+ cells, or to a previously reported DNA methylation profile in a separate cohort of 344 AML patients39. Unbiased motif analysis of differentially methylated gene promoters showed an enrichment of the motif recognized by the N-terminal ZF domain of EVI139. DNMT3A was also found to be highly expressed in EVI1-high AML samples compared to other AML subtypes. EVI1 expression levels correlated positively with a stronger hypermethylation signature in AML patient samples39.EVI1 interacts through its N-terminal ZF domain with the de novo DNA methyltransferases DNMT3A and 3B40. This region was also critical for EVI1 repression of TGF-β (transforming growth factor beta) signaling and was thus termed the repressive domain (Rp) , a subunit of the transcription factor AP-1. Mutation of the 6th and 7th EVI1 zinc fingers mitigated EVI1 interaction with PU.1 and restored differentiation potential to 32Dcl3 cells in response to G-CSF34. EVI1 interaction with AP-1 subunits c-Fos and c-Jun was noticed as early as 1994 by Tanaka et al.44. EVI1-expressing cells exhibited increased c-Fos and c-Jun levels, and the C-terminal ZF domain of EVI1 was critical for activation of the c-Fos promoter44. Loss of EVI1 decreased c-Fos occupancy on the DNA, suggesting that EVI1 and AP-1 may act cooperatively at some loci6. A SILAC-MS screen also confirmed c-Fos and c-Jun interaction with EVI129. This screen also identified several additional transcription factors and co-factors that interact with EVI1, and 65% of EVI1-regulated genes were upregulated29. This highlighted the role of EVI1 as a transcriptional activator.A number of gene targets are upregulated by EVI1 Table . EVI1 in45 /t rearrangements can also be observed in up to 25–40% of CML patients in blast crisis49. Despite their existence as distinct clinical entities, MDS, AML, and CML with inv(3)/t rearrangements have similar cytogenetic abnormalities, molecular alterations, pathological features, and poor prognosis52. In inv,(3) breaks most frequently occur in a region between RPN1 (Ribophorin 1) and C3orf27, downstream of GATA2, that contains a distal GATA2 hematopoietic enhancer  and the region between C3orf50 and the first exon of the MECOM locus that encodes for the MDS1-EVI1 transcript , the breakpoint frequently occurs in between the MDS1 promoter and the first EVI1 exon, and MDS1-EVI1 transcript is frequently lost /t AML54. The new enhancer region generated by the chromosomal rearrangement was noted to contain a 9-kb region with a p300 binding site that interacts with the EVI1 promoter, and removal of this binding site attenuates EVI1 expression53. Transgenic mice in which the human inv(3) chromosomal abnormality was recapitulated through a bacterial artificial chromosome developed leukemia, but if the relocated GATA2 enhancer region was deleted, EVI1 expression declined and leukemia did not develop54. These seminal studies confirmed that EVI1 dysregulation in response to chromosomal rearrangements in myeloid disease occurred not from the typical generation of a fusion transcript, but rather from “enhancer hijacking”54. This highlighted a two-fold impact, one leading to overexpression of EVI1 and the second causing haploinsufficiency of GATA2, as it is no longer expressed in the rearranged chromosome53.In the World Health Organization (WHO) classification of AML and related neoplasms, inversion or translocation of chromosome 3 at the MECOM locus  have been recognized as recurrent genetic abnormalities55. Most atypical 3q26 rearrangements have levels of EVI1 overexpression comparable to inv(3)/t cases, similar phenotypic changes, and share a poor prognosis55. Atypical 3q26 rearrangements include, but are not limited to t, t, t, and t, which involve THADA (THADA armadillo repeat containing), CDK6 (cyclin dependent kinase 6), MYC , and ARID1B (AT-rich interaction domain 1B), respectively55. Similar to the “enhancer hijacking” of the GATA2 distal hematopoietic enhancer in inv(3)/t, atypical 3q26 rearrangements also seem to overexpress EVI1 through repurposing of enhancer elements from the translocation partners. In ten cases of atypical 3q26 rearrangements, EVI1 was overexpressed and the translocation partner whose enhancer was repurposed had decreased expression, with the exception of MYC in t55.In ~0.5–1% of AML and MDS patients, atypical chromosome 3 rearrangements occur involving the MECOM locus56  breakpoints are between the ETV6 exon 2 and 3 and on heterogeneous regions in 3q26, both 3′ and 5′ of MDS1 as well as in between MDS1 and EVI156. The resulting translocation fuses the first two exons of ETV6 with the entire MDS1-EVI1 or EVI1 transcript. Since no known functional domain of ETV6 is added to EVI1 in the fusion protein, it is thought that the oncogenic properties of the fusion protein come from the inappropriate expression and function of EVI1 driven by the ETV6 promoter56. In line with this, similar to other 3q26 rearrangements, myeloid malignancies expressing the ETV6-EVI1 fusion are associated with dysmegakaryopoiesis and poor prognosis58. The t can generate RUNX1-MDS1-EVI1 fusion protein, where the DNA-binding RUNT domain of RUNX1 is fused to the whole MDS1-EVI1 protein  and tq26;q24) that result in ETS variant transcription factor 6 -EVI1 and RUNX1-MDS1-EVI1 fusion proteins, respectively that res61. Several ChIP studies have shown that mixed lineage leukemia (MLL) and MLL fusion proteins, including MLL-AF9 and MLL-ENL bind to the EVI1 regulatory region, resulting in increased EVI1 expression63. In a recent report in which MLL-AF9 fusion gene was expressed either in murine Sca−Kit+ (LSK) HSCs or in granulocyte monocyte precursors (GMPs), LSK-MLL-AF9 cells had significantly higher levels of Evi1 than GMP-MLL-AF9 cells64. Additionally, AMLs with high EVI1 expression have been shown to be associated with inferior relapse-free and overall survival65.Aberrant EVI1 expression can also occur in the absence of chromosome 3 rearrangements. EVI1 overexpression is observed in ~8–10% of MDS, 8% of de novo AML, and 30% of advanced CML, but it is unclear here how EVI1 overexpression occurs29. Furthermore, the EVI1 N-terminal ZF domain binds to GATA-like SSRs, and EVI1 ChIP analysis revealed an increase in recombination rates near EVI1 bound SSR24. How EVI1 increases genomic instability is not well characterized beyond its protein interactions. However, a gene therapy study using a Maloney murine leukemia virus vector to express NADPH-oxidase conducted in two patients to treat chronic granulomatous disease unfortunately caused integration of the vector at the MECOM locus. The patients developed clonal expansion of myeloid cells bearing activating insertions in the MECOM locus and EVI1 overexpression. Both patients developed monosomy 7 in the dominate clone, suggesting that EVI1 could favor expansion of clones with monosomy 7 or that EVI1 could contribute to the genomic instability leading to monosomy 713.Utilizing SILAC-MS studies to determine EVI1 interaction partners, Bard-Chapeau et al. observed enrichment in protein domains associated with DNA repair, chromatin remodeling, and transcription43. Enforced Evi1 expression transcriptionally repressed C/EBP-α in murine hematopoietic cells18. EVI1-mediated repression of C/EBP-α was also observed in the murine hematopoietic progenitor cell line 32Dcl317. Further confirmation that EVI1 represses C/EBP family members is needed through in vivo leukemia models and in patient-derived samples. EVI1 also regulates hematopoietic differentiation and proliferation through transcriptional repression of several miRNAs. EVI1 repressed miR-9 levels through binding to its regulatory region, recruiting DNMT3B, and inducing DNA methylation66. Decreased miR-9 led to increased levels of its target genes FOXO1 and 3 (Forkhead Box O1 and 3)66. EVI1 expression was also found to decrease miR-449A levels, and ChIP analysis showed EVI1 bound miR-449A regulatory region67. Repression of miR-449A by EVI1 increased expression of the miR-449A-targets Notch1 and Bcl-2 in human AML cell lines67.EVI1 is known to directly interact with and repress the activity of a number of myeloid transcription factors including GATA1, PU.1, and RUNX168. Knockdown of PBX1 decreased EVI1-mediated transformation of primary mouse bone marrow cells68. Comparing tissues from wild type to those from EVI1+/− and EVI1−/− mice, at embryonic day 9.5, GATA2 expression was decreased in EVI1 depleted tissues69. EVI1 expression also correlated with high expression of megakaryocytic markers, including the thrombopoietin receptor MPL70. Furthermore, in a mouse model of EVI1 leukemia, thrombopoietin expression correlated with EVI1 expression, and double positive EVI1-thrombopoietin cells had enhanced secondary leukemia formation ability in a serial bone marrow transplant assay70. Collectively, in myeloid malignancies expressing EVI1, transcriptional alterations of specific myeloid transcription factors, and of miRNAs, contribute to myeloid dysplasia.EVI1 transcriptionally activates the hematopoietic proto-oncogene PBX1 (PBX homeobox 1) through binding to its promoter region33. Conversely, knockdown of EVI1 was shown to decrease Bcl-xL levels by approximately five fold33. EVI1 interactions with the microenvironment are also implicated in apoptosis-resistance. The adhesion molecules ITGA6  and GPR56 (adhesion G protein-coupled receptor G1) are highly expressed in EVI1-positive AML, and their knockdown leads to increased apoptosis in response to Ara-C treatment and loss of RhoA (ras homolog family member A) signaling, respectively72. In AML, cells high EVI1 expression correlated with high MYC and BCL2 expression, with poorer clinical outcome73.Several pathways have been implicated in EVI1-mediated resistance to apoptosis leading to drug-resistance. High EVI1 expression correlated with high expression of the anti-apoptotic Bcl-xL protein in CML patient samples51. Categorized as high risk, more than half of inv(3)/t MDS patients with EVI1 overexpression progress to AML within ~2 years of diagnosis47. Furthermore, EVI1 overexpression correlates with shorter overall survival and poorer response to treatment51. Overall survival of patients with EVI1-positive MDS ranges from 13 to 17 months after diagnosis47. Like EVI1-positive MDS, AML with EVI1 overexpression often presents with myeloid dysplasia, particularly of the erythrocyte and megakaryocytic lineages51. Studies have also reported EVI1 expression as an independent prognostic factor for poorer overall survival in AML, and high EVI1 expression is associated with poorer response to therapy75. Several clinical studies have reported that, in 3q26-rearranged AML, the median overall survival after diagnosis remains approximately less than 1 year, whereas long-term overall survival is less than 15%  M51. EVI1 expression is rarely detected in the chronic phase of CML, but is readily detected in a significant proportion (25–40%) of blast crisis of CML, suggesting that acquisition of EVI1 expression can drive progression into blast crisis49. EVI1 expression in CML blast crisis is correlated with poor response to therapy, and has been linked with acquisition of resistance to tyrosine kinase inhibitors48.EVI1-expressing CML may also be associated with megakaryocytic dysplasia74. Retrospective studies have shown that inv(3)/t MDS/AML with −7/−7q display worse prognosis than inv(3)/t alone77. Which genetic alteration occurs first is unclear, and likely varies on a case-by-case basis, given the heterogeneity of the myeloid malignancies. As noted above, in two cases where gene therapy activated EVI1 expression through retroviral insertion, both cases developed monosomy 7 in the dominant leukemic clone, suggesting that EVI1 at least favors events leading to monosomy 713. The q arm of chromosome 7 contains several key genes whose haploinsufficiency is considered to be a loss of tumor-suppressor and thus contribute to leukemia transformation. These genes include EZH2 and MLL3, as well as the cytoplasmic cellular regulators SAMD9 (Sterile Alpha Motif Domain Containing 9) and SAMD9L78. Perhaps due to the ability of MLL and MLL-fusion proteins to upregulate EVI1 transcription, EVI1 overexpression can be observed in ~30% of cases with MLL translocation, and here EVI1 expression correlates with poor prognosis65.Loss of one copy of chromosome 7  or deletion of the long arm of chromosome 7 (−7q) is observed in 30–70% of MDS and AML with inv(3)/t79. Another transcription factor IKZF1 (IKAROS family zinc finger) is also mutated in up to 25% of cases of inv(3)/t MDS or AML. Since IKZF1 is located on chromosome 7, IKZF1 mutations occur in clones without chromosome 7 deletions77. Although not a mutation, almost all MDS, AML, and CML with inv(3)/t have GATA2 haploinsufficiency due to the re-location of the GATA2 distal hematopoietic enhancer54. This was shown to contribute to EVI1-driven leukemia transformation21. Of note, despite loss of expression from one allele of GATA2, 15% of inv(3)/t can carry additional mutations in GATA2 on the non-rearranged allele77.Approximately 20% of MDS and AML patients with inv(3)/t express mutations in RUNX179. These mutations are observed in 66–98% of inv(3)/t MDS/AML79. A greater percentage of AML cases with inv(3)/t AML carried RAS family mutations, as compared to the MDS cases79.A significant proportion of inv(3)/t MDS and AML cases have activating mutations in RAS GTPase family member (NRAS or KRAS), or in other RAS-signaling pathway proteins, including PTPN11 (protein tyrosine phosphatase non-receptor type 11), and NF1 (neurofibromin 1), which promote dysregulated RAS signaling and uncontrolled proliferation77. However, mutations in the polycomb group protein ASXL1  were reported in ~20% of AML cases with inv(3)/t77. Mutations in splicing factors SF3B1 (splicing factor 3b subunit 1) and U2AF1  were also found in ~30–60% of inv(3)/t MDS or AML cases77. The biologic impact of these ‘epimutations’ in myeloid malignancies on the transcriptional signature attributed to inv(3)/t and EVI1 overexpression remains to be elucidated.Low frequency of mutations in DNMT3, TET2 (tet methylcytosine dioxygenase 2), and IDH1/2 (isocitrate dehydrogenase 1/2) were observed in AML or MDS with inv(3)/t79. Why EVI1 overexpression is not seen with NPM1 or C/EBP-α mutations is unknown. One possibility could be that survival of the clones with high EVI1 expression in combination with a NPM1 or C/EBP-α mutation is impaired.Mutations in NPM1 (nucleophosmin 1) and C/EBP-α inversely correlate with inv(3)/t and EVI1 expression74. In a study by the Delwel group, allogenic hematopoietic stem cell transplant following the first clinical remission yielded increased survival odds in AML patients with MLL translocation with EVI1 overexpression65. However, greater than 40% of the patients still succumbed to their disease65. Overall survival rate of patients with CML who initially respond but later progress on TKI therapy and acquire EVI1 overexpression is half compared to those without EVI1 expression48.Standard front-line treatment of MDS includes DNA de-methylating agents like azacitidine and decitabine. In advanced, high-risk MDS carrying inv(3)/t with an increased percent of bone marrow blasts between 5 and 20%, or in overt transformation of MDS to secondary AML (sAML), chemotherapy with cytarabine (Ara-C) and the anthracyclines, idarubicin and daunorubicin is commonly employed. In CML, treatment in the chronic phase generally begins with a tyrosine kinase inhibitor (TKI) such as imatinib, dasatinib or nilotinib. In transformation of CML into blast crisis (CML-BC) carrying inv(3)/t or t, therapy with a second generation tyrosine kinase inhibitors and/or chemotherapy is utilized. EVI1-positive myeloid malignancies have been documented to be relatively refractory to current therapies. There is no statistical difference in the overall 5-year survival rates between MDS and AML with inv(3)/t, which averages at 3–5%80. A targeted agent has yet to be identified and developed that exhibits clinical efficacy specifically against EVI1-overexpressing myeloid malignancies.To date, following treatment of myeloid malignancies with inv(3)/t or EVI1 overexpression with targeted therapies, including DNA hypomethylating drugs, venetoclax or glasdegib, or with FLT3 TKI or IDH1/2 inhibitors, clinical outcome data are unavailable81. By also inhibiting GATA2 super enhancer, treatment with BET  inhibitor could repress EVI1, as well as reverse EVI1-dependent transcriptional programs through inactivating enhancers and super enhancers of the key oncogenes /t and/or EVI1 overexpression53.One promising target is the chromatin reader protein BRD4 (bromodomain containing 4), which is involved in transcriptional activation, especially via sustaining the activity of super enhancers, such as those of MYC, CDK4/6 and BCL2/Bcl-xLnes Fig. . Preclin32. For example, RUNX1 interacts with EVI1 and is positively regulated by acetylation84. Furthermore, the GATA2 distal enhancer that is relocated and transactivates EVI1 in inv(3) and t chromosomal aberrations contains a p300 binding site that is critical in driving EVI1 expression54. Additionally, the GATA2 enhancer has increased read-through of enhancer RNAs (eRNAs) at the breakpoints that cause its repositioning, and the CBP/p300 inhibitor GNE-049 is reported to particularly inhibit eRNAs85. Therefore, targeted combination therapy with this HAT inhibitor would simultaneously cause deacetylation of the transcription factors, leading to decreased transcriptional activity, as well as cause the loss of super enhancer function, thereby effectively shutting down EVI1 transcriptional program.Several transcriptional regulators including EVI1 are acetylated by CBP/p30086. EVI1 expression is also associated with hypermethylation of over 200 genes in AML samples86. Consistent with this, DNA methyltransferase inhibitors have exhibited clinical activity in EVI1-overexpressing AML87. However, since monotherapy with DNA hypomethylating agent as a first-line treatment for MDS exhibits only a modest clinical efficacy, combination with other targeted therapies is likely to achieve superior efficacy against EVI1-expressing MDS. Use of EZH2 inhibitor to abrogate dependency of EVI1-expressing MDS or AML with monosomy 7 on the residual normal EZH2 function may exert added efficacy.EVI1 can repress transcription through recruitment of DNMT3A or B resulting in de novo methylation88. EVI1-mediated upregulation of the anti-apoptotic Bcl-xL suggests that BH3-mimetic inhibitor targeting this anti-apoptotic protein could also have therapeutic value, alone or in combinations against EVI1-expressing myeloid malignancies. Recently, the small molecule compound pyrrole-imidazole polyamide, which inhibits the DNA-binding activity of N-terminal ZF domain of EVI1, was shown to induce apoptosis of EVI1-expressing AML cells due to downregulation of the EVI1 target GRP56, which was linked to EVI1-mediated resistance to apoptosis71. Several therapies targeting anti-apoptotic proteins are in clinical trials, and these agents could potentially be employed in treatment of EVI1-expressing myeloid malignancies.Recently, co-treatment with venetoclax, a BH3-mimetic inhibitor of Bcl-2 protein, with azacitidine was approved for therapy of AML28. Therefore, along with treatment with BCR-ABL1 targeted TKI inhibition of β-catenin/TCF signaling may be a promising strategy. Additionally, several groups have demonstrated that resistance to BET inhibitors in AML is mediated by the activity of β-catenin/TCF7L2/c-Myc axis, resulting in re-expression of c-Myc despite treatment with BET inhibitor89. In this setting also, treatment with a β-catenin/TCF signaling inhibitor, e.g., BC2059 (tegavivint), may not only reverse BET inhibitor resistance but also exhibit synergy with BET inhibitor against AML with inv(3)/t and/or EVI1 overexpression90.In CML with inv(3)/t, β-catenin/TCF1 (T cell factor 1) signaling was reported to be activated, which positively regulated EVI1While great strides have been made, there is still much more to be elucidated regarding EVI1 biology and its contributions to leukemogenesis. EVI1 is a transcriptional regulator that promotes a stem-like expression program in hematopoietic progenitors, crucial to their self-renewal, growth, and repopulating potential. The role of co-expression of alternative EVI1 transcripts in myeloid malignancies remains to be determined. EVI1 regulates transcription through recruitment of epigenetic modifiers. Recent studies have highlighted the dysregulated transcriptome and signaling pathways that underpin the aberrant biology, aggressive phenotype, and refractoriness to standard therapy of EVI1-overexpressing myeloid malignancies Fig. . How com