The human Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is a severe disease with increased mortality caused by mutation in the LSH gene. Although LSH belongs to a family of chromatin remodeling proteins, it remains unknown how LSH mediates its function on chromatin in vivo. Here, we use chemical-induced proximity to rapidly recruit LSH to an engineered locus and find that LSH specifically induces macroH2A1.2 and macroH2A2 deposition in an ATP-dependent manner. Tethering of LSH induces transcriptional repression and silencing is dependent on macroH2A deposition. Loss of LSH decreases macroH2A enrichment at repeat sequences and results in transcriptional reactivation. Likewise, reduction of macroH2A by siRNA interference mimicks transcriptional reactivation. ChIP-seq analysis confirmed that LSH is a major regulator of genome-wide macroH2A distribution. Tethering of ICF4 mutations fails to induce macroH2A deposition and ICF4 patient cells display reduced macroH2A deposition and transcriptional reactivation supporting a pathogenic role for altered marcoH2A deposition. We propose that LSH is a major chromatin modulator of the histone variant macroH2A and that its ability to insert marcoH2A into chromatin and transcriptionally silence is disturbed in the ICF4 syndrome. The human ICF 4 syndrome is caused by mutation of the chromatin remodeller LSH. Here, the authors show that LSH depletion disrupts the ability of histone variant macroH2A to insert into chromatin and silence transcription. The octamer is composed of a central tetramer of histones H3 and H4 flanked by two heterodimers of histones H2A and H2B. Chromatin dynamics is controlled by histone modifiers and chromatin remodeling complexes that alter chromatin structure and regulate nuclear processes such as transcription, replication, recombination, or DNA repair5.Eukaryotic chromatin is built of arrays of nucleosomes that each consists of 147 bp DNA wrapped around an octamer of core histones6. Apart from a linker and a macrodomain, macroH2A contains a histone domain that is ~65% identical to canonical H2A, but differs in the L1 loops which is important to stabilize DNA binding to the octamer7. Reconstitution of nucleosomes containing macroH2A renders them more resistant to exonuclease digestion and promotes interactions between nucleosomes8. This furthers chromatin condensation and contributes to the more compacted nature of heterochromatin9. MacroH2A is a family of variants, including macroH2A1.1 and macroH2A1.2 (isoforms transcribed from one gene) and macroH2A2, which are frequently associated with constitutive or facultative heterochromatin and transcriptional silencing12. However, macroH2A1 can also reside in euchromatin and positively influence transcription, a feature which is primarily attributed to the isoform macroH2A1.114. Recently, it has been suggested that macroH2A may reinforce active or repressed expression states and buffer transcriptional noise15. Several factors have been identified that negatively regulate macroH2A incorporation in vivo, however, factors that control its deposition into chromatin remain unknown9.The canonical core histones can be replaced with histone variants, including macroH2A17. The pathophysiology of ICF4 remains largely unknown, but the autosomal recessive disease is caused by a genetic mutation in LSH/HELLS . Lsh knockout mice phenocopy many symptoms, and exhibit hematopoietic, neurologic, and germ cell deficiencies, reduced growth, and early lethality23.Patients with Immunodeficiency, Centromeric instability and Facial dysmorphism syndrome type 4 (ICF4) suffer from a plethora of symptoms including organ malformations, facial anomalies, mental retardation, delayed growth, genomic instability, and severe immunodeficiency which leads to a shortened lifespan25. Members of the family are thought to translocate along DNA. This characteristic enables them to evict nucleosomes, slide nucleosomes along DNA or exchange histone variants. LSH can perform nucleosome sliding in vitro and its activity is enhanced by CDCA7, a protein that causes a related syndrome, known as ICF2, when it is mutated28. LSH recruitment results in reduced chromatin accessibility in vivo and LSH function is associated with transcriptional silencing31. LSH exerts its effects on repeat sequences embedded in heterochromatin and is associated with high DNA methylation levels32.LSH belongs to the SNF2 family of ATP-dependent chromatin remodelers33. We define a role of LSH in macroH2A deposition and examine its potential role in ICF4 patients.While LSH function is associated with heterochromatin it remains unknown if and how it induces repressed chromatin states and whether this relates to its chromatin remodeling function. Here, we delineate the causal sequences of biochemical events induced by LSH using chemical induced proximityOct4 allele34. We utilized a previously generated murine embryonic stem (ES) cell line containing an array of DNA binding domains (12 × ZFHD1) upstream of the Oct4 transcription start site (TSS) on one allele . Furthermore, the DNA binding domain of ZFHD1 was stably expressed as a fusion protein with the rapamycin binding domain FKBP-HA tagged (ZnF-FKBP-HA). Addition of rapamycin to the cell culture elicits an interaction between the two fusion proteins and recruitment of the complex to the modified Oct4 allele35. In contrast to direct fusion of a protein to the DNA binding domain, which produces a rigid conformation, the fast off rate of rapamycin allows for more freedom of activity and a normal mode of action, as has been shown for the BAF complex36.To define the primary molecular function of LSH, we applied a chemical-induced proximity (CIP) assay in which a fusion protein can be tethered to one genetically modified ele Fig. 34,35. InOct4 allele within 15–60 min as measured by chromatin immunoprecipitation (ChIP) followed by qPCR analysis  within its ATP binding site. This site is critical for ATP hydrolysis and chromatin remodeling activity of SNF2 like factorsLSH Fig.  and addicus Fig. . Remarkacus Fig. , suggestOct4 allele . We used EFs Fig. 12,49, sised Fig. . Likewissed Fig. . Moreoveons Fig. . To testTo investigate whether LSH mediated transcriptional silencing depends on macroH2A1.2 or macroH2A2 deposition, we depleted macroH2A in the genetically modified ES cells by siRNA interference Fig. . After s50. Therefore, we analyzed repeat sequences that exhibited a decrease in macroH2A enrichment after LSH depletion  ES cells compared to wild-type ES cells  expression vector . Flagged-tagged LSH readily pulled down macroH2A1 as well as macroH2A2, but less so canonical H2A Fig. . FurtherLsh−/− and Lsh+/+ MEFs that express predominantly macroH2A1.2 and macroH2A2  comparing macroH2A1 to macroH2A2 patterns in wild-type cells  and R = 0.680 (log2 transformed data) indicating moderate to strong association between LSH occupancy and macroH2A deposition in the genome  showed significantly greater differences of macroH2A occupancy in LSH-deficient cells compared to the average gene, while H2B enrichment showed no differences  patientent Fig. . Westernent Fig. . Using Cent Fig. . Furtherent Fig. . Correspent Fig.  comparab9. For example, ATRX inhibits macroH2A accumulation at telomeres and at the alpha globin locus, and FACT removes macroH2A from transcriptionally active regions59. Our study suggests that LSH promotes incorporation of macroH2A into chromatin for part of the genome. We show here that tethered LSH induces macroH2A1.2 and macroH2A2 accumulation at the recruitment site and that H2A is reciprocally reduced. In addition, the ATP binding site of LSH is required for macroH2A1.2 and macroH2A2 deposition implying chromatin remodeling activity of LSH in the process. LSH can interact with macroH2A, and this interaction shows some specificity, since two ICF4 mutant proteins and the histone variant H2A.Z do not show the same degree of interaction. Furthermore, under physiologic conditions, LSH is critical for the genome wide establishment of broad macroH2A domains including heterochromatin containing repeat sequences and ribosomal genes. Thus, LSH is either directly involved as a histone exchange factor or indirectly, coordinating the exchange reaction.A few factors have been found to remove macroH2A from its genomic location, but no deposition machinery has been identified which is involved in genome wide incorporation64. Most chromatin remodelers can use their ATP-dependent DNA translocase activity to slide nucleosomes along DNA, but histone replacement can also occur without repositioning of nucleosomes24. Some factors such as INO80C can slide nucleosomes, as well as perform histone exchange. Notably, LSH and its binding partner CDCA7 can increase nucleosome mobility in vitro in a restriction enzyme accessibility assay26. Interestingly, we observed an asymmetrical deposition of macroH2A1.2 and macroH2A2 with respect to LSH binding. Most remodelers anchor at a fixed position on the histone octamer and show unidirectional sliding along DNA24. The recruitment of LSH at the genetically modified Oct4 locus may give LSH a certain orientation and the translocase domain of LSH may impose unidirectional movement along DNA, which facilitates H2A-H2B removal and subsequent marcoH2A-H2B deposition. Known chromatin remodelers that act as nucleosome editors include SWR1 and p400 that replace H2A-H2B dimers with H2A.Z, INO80C which performs the reverse reaction or can remove the histone variant H2A.X and p400 that replaces H3.1 with the variant H3.324. Our data demonstrates that LSH is a critical factor in genome wide macroH2A deposition. However, several observations suggest that LSH is not the only factor and redundancy may exist. For example, we observed ~30–50% reduction in broad domains and peaks consistent with the ~40% decrease in chromatin association comparing LSH deficient cells to wild-type controls. This indicates a widespread effect of LSH on genomic macroH2A distribution, however, some macroH2A was still deposited in the absence of LSH. In addition, the combination of macroH2A and LSH deletion revealed that macroH2A deletion had an additive effect on LSH deletion in terms of transcription and nuclear size, while LSH deletion did not exacerbate macroH2A deletion. This suggests that, LSH may mediate most of its effect on transcription and nuclear size via macroH2A, but that other so far unknown factors may also be capable to promote macroH2A deposition or may be able to substitute for LSH.Chromatin remodeling enzymes of the SNF2/Rad54 helicase family, to which LSH belongs, are evolutionarily conserved and can participate in chromatin assembly, repositioning of nucleosomes or exchange of histone variants12. However, the pruning mechanism alone may not explain why low levels of macoH2A are found at gene-poor sites61 and how macroH2A enrichment can be decreased in LSH-deficient cells at sites that do not show any transcriptional changes. Thus, our model suggests that multiple pathways, including a LSH dependent pathway and the transcriptional pruning pathway via the histone chaperone FACT12, act together to shape genome wide macroH2A deposition. Our model proposes that LSH deposits macroH2A at specific regions of the genome, and transcription prunes and fine tunes the amount of macroH2A at any given transcriptionally active genomic site.A recent report has shown that transcription is an important mechanism to remove macroH2A after it has been deposited across the genome by yet unknown factor(s)Hox cluster target genes66; removal of macroH2A increases the efficiency of induced pluripotency67 and LSH removal enhances cellular plasticity68; knockdown of macroH2A or LSH in mouse ES cells results in incomplete silencing of pluripotency genes Oct4, Nanog, and Sox2 after differentiation70; knockout of macroH2A or LSH in mice results in smaller body size of day18.5 fetus and mice show increased perinatal lethality (30% and 100% respectively), but neither phenotype shows a pathology that would point to the cause of death71. Protein coding genes display only modest deregulation in either macroH2A or LSH-deficient cells, however, diverse repeat elements and retroviral elements are transcriptionally de-repressed53. There are reproductive problems in macroH2A knockout mice in C57BL/6 background and Lsh knockout mice show defects in male and female germ cell development72. While macroH2A knockout mice share some phenotypic similarities with Lsh knockout mice, not all LSH effects maybe mediated by macroH2A. For example, Lsh knockout mice exhibit more severe lethality and LSH-deficient cells still have a remainder of macroH2A deposition, suggesting that they do not share a completely congruent pathway.If some effects of LSH were mediated via macroH2A deposition, we would expect that LSH and macroH2A depletion show a likeness in their phenotypes. There is some degree of similarity, for example, macroH2A or LSH depletion reduces polycomb repression at 73 and the nature of the mutants’ malfunction maybe manifold. We observed reduced interaction of two ICF4 mutants with macroH2A which may explain their impaired ability to deposit macroH2A. Other potential malfunctions of ICF4 mutant proteins may include the ability to perform DNA translocation, to mobilize the H2A-H2B dimers or to interact with other factors of a presumed LSH complex that may be critical for macroH2A-H2B dimer recognition and the exchange reaction73. ICF4 (L 801 del) patient cells lack normal macroH2A accumulation at several repeat sequences and fail to repress those loci, indicating that ICF4 patients suffer from abnormal histone variant deposition. To our knowledge, macroH2A knockout mice have not yet been assessed for some of the hallmarks of the ICF4 syndrome such as immunoglobulin deficiency, genomic instability and neurologic malfunction. Several genetic diseases have been uncovered, involving mutations in a chromatin remodeler, chaperone, or histone variant, but no disease has been as yet attributed to abnormal macroH2A deposition75. MacroH2A as well as LSH has been implicated in processes of transcriptional regulation, DNA repair, genomic stability, and tumor development78 and it will be important in the future to discern which molecular pathways partially or completely overlap and contribute to the pathophysiology of the ICF4 syndrome.Importantly, we also found that ICF4 mutations fail to incorporate macroH2A at tethered sites and show impaired capacity for transcriptional silencing. Histone exchange is a multi-step processCiA: Oct4 mouse ES cell line34 contains one modified Oct4 allele harboring one array of DNA binding sites (12 × ZFHD1) in the promoter region upstream of an in-frame nuclear eGFP reporter inserted at the ATG of exon 1 of Oct4. WT (Lsh+/+) and KO (Lsh−/−) mouse ES cell lines had been derived from mouse embryos, and KO ES cells had been transfected with Lsh WT or Lsh mutant (K 237A) expression vector27. All mouse ES cells were grown on 0.1% gelatin (Sigma) coated six-well plates in KnockOutTM DMEM (Gibco) media supplemented with 15% KnockOut™ Serum Replacement (Gibco), 10 mM HEPES pH 7.5 (Gibco), 1× Minimum Essential Media (MEM) nonessential amino acids (Gibco), 1× GlutaMAXTM (Gibco), 50 U ml−1 Pen/Strep (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), and 1:10,000 ESGRO® Recombinant Mouse LIF Protein (Millipore). WT (Lsh+/+) and KO (Lsh−/−) MEF cell lines30 were grown in high-glucose DMEM (Invitrogen) supplemented with 10% Fetal Bovine Serum , 1× GlutaMAXTM and 50 U ml−1 Pen/Strep. U2OS (ATCC), HEK293T (ATCC), and normal human lung fibroblast  cell lines were cultured in high-glucose DMEM (Invitrogen) supplemented with 10% FBS and 50 U ml−1 Pen/Strep. ICF4 parent and patient cell lines 17 were grown in RPMI-1640 (Gibco) media supplemented with 20% heat-inactivated FBS (Omega Scientific), 1× GlutaMAXTM and 50 U ml−1 Pen/Strep. All cells were cultured at 37 °C in a humidified incubator containing 5% CO2.The 79. Concentrated lentiviral supernatants by Lenti-XTM Concentrator (Takara) were used to infect CiA: Oct4 mouse ES cells with 8 μg ml−1 polybrene (Sigma). Cells were incubated overnight prior to virus removal and selection of lentiviral construct achieved in CiA: Oct4 with either: ZnF-FKBP-HA plasmid, puromycin (2 μg ml−1) or LSH (WT or Mutant)-FRB-V5 plasmid, hygromycin (400 μg ml−1). ZnF-FKBP-HA fusion protein was directly recruited to the indicated DNA binding domains (12 × ZFHD1) and used to harbor LSH (WT or Mutant)-FRB-V5 fusion protein. Proximity of FRB and FKBP was induced by addition of rapamycin (Selleckchem) at 3 nM in all experiments. Lsh MISSION shRNA-expressing lentiviral vector was from Sigma. After lentivirus production and infection, U2OS cells were selected with puromycin (2 μg ml−1) to generate stable cell line. Transient transfections of U2OS cells with 3 × FLAG-LSH (WT or ICF4-Mutant)-IRES2-GFP, macroH2A2-GFP, and GFP vectors were performed by using TurboFect transfection reagent (Invitrogen). Afterwards, two continuous rounds of fluorescence-activated cell sorting (FACS) were utilized to collect respective GFP positive U2OS cells in 3 and 15 days in order to generate stable cell line. macroH2A1 and macroH2A2 siRNAs  were transfected using Lipofectamine RNAiMAX transfection reagent (Invitrogen) following the manufacturer’s instructions and analyzed 48–72 h post transfection.Lentivirus was produced by Lipofectamine 2000 (Invitrogen) transfection of HEK293T cells with gene delivery vector co-transfected with packaging vectors pspax2 (Addgene) and pMD2.G (Addgene)All flow cytometry analyses were performed on a LSR II (BD Biosciences) and analyzed with FlowJo v. 10 software. Cell population of each sample was gated using unmodified mouse ES cells without GFP expression as negative controls. Individual cells were gated based on forward and side scatter, autofluorescent cells were omitted, and remaining cells were analyzed for GFP level.Gapdh abundance. Primers are listed in Supplementary Table Total RNA was isolated using Rneasy plus Mini kit (Qiagen) following with additional RNase-free DNase treatment. Reverse transcription was performed by using PrimeScript RT Reagent Kit (Takara). Transcripts were amplified using MyiQ2 two colors Real-Time PCR detection system (Bio-rad) with iTaq Universal qPCR Mastermix (Bio-rad). Relative expression was normalized to internal 80 1 × 107 cells were cross-linked with 1% formaldehyde, lysed, and sonicated on ice to generate 200–800 bp DNA fragment. In all, 1% of each sample was saved as input fraction. Immunoprecipitation was performed using chip-grade antibodies listed in Supplementary Table For ChIP assays81. Primers for PCR amplification are listed in Supplementary Table Genomic DNA was extracted using the DNeasy blood & tissue kit (Qiagen). For each reaction, 1 μg genomic DNA was bisulfite converted with the EpiTec Bisulfite Kit (Qiagen). Knock-in-specific eGFP sequence was amplified by PCR. The PCR products were cloned by using TOPO TA cloning kit (Invitrogen). IAP repetitive element was used as a positive control. After screening of positive clones and digestion of plasmids with EcoRI (NEB), 10 clones for each sample were sequenced to identify the methylation level of cytosine. Methylation profiles were analyzed by using BiQ Analyzer version 0.7 software82, buffers were supplemented with 0.5 mM dithiothreitol (DTT), 0.1 mM phenylmethyl sulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail. About 1 × 107 cells were resuspend cells in 1 ml buffer A  and incubated on ice for 10 min. Nuclei were isolated by centrifugation at 1500×g, 4 °C. The supernatant was taken as cytosolic fraction. Nuclei were washed once with buffer A and lysed for 30 min in buffer B (3 mM EDTA and 0.2 mM EGTA) on ice. Chromatin fraction was pelleted by centrifugation at 1500×g, 4 °C. Whole-cell extract were prepared using 2 × 106 cells. Cells were lysed on ice for 15 min with 150 μl RIPA buffer . The lysate was sonicated for 30 s with 50% pulse and centrifuged for 15 min at 14,000×g, 4 °C. The supernatant was collected, and protein concentration was measured by bicinchoninic acid assay (BCA) assay (Thermo Fisher). Samples were boiled in Laemmli sample buffer (Bio-Rad) at 95 °C for 5 min. Equal amounts of protein were loaded onto acrylamide/bis gels and transferred to PVDF membranes after electrophoresis. Following blocking in 5% nonfat milk for 1 h, membranes were incubated at 4 °C overnight in primary antibodies listed in Supplementary Table For cell chromatin fractionation7 U2OS cells expressing 3× FLAG-LSH (WT or ICF4-Mutant) and macroH2A2-GFP were collected and washed twice with PBS. Cell pellets were resuspended with IP lysis buffer  supplemented with fresh 0.5 mM DTT, 0.1 mM PMSF, and 1× protease inhibitor cocktail. Incubate cell lysates on ice for 10 min with periodic mixing. Remove cell debris by centrifugation at 13,000×g for 10 min at 4 °C. The supernatant was collected and protein concentration was measured by BCA assay. For each IP, 1 mg protein was diluted with IP lysis buffer to 1 ml, and 50 μl was taken as input. Precleaning was performed by addition of 50 μl Protein G Magnetic Beads (Bio-rad) and incubation for 1 h rotation at 4 °C. Each precleaned sample was mixed with 4 μg antibody respectively listed in Supplementary Table For CO-IP analysis 2 × 10Cells grown on chamber slides were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked in 5% BSA for 30 min, then incubated at 4 °C overnight in primary antibodies listed in Supplementary Table 83 all buffers were freshly supplemented with 0.5 mM DTT, 0.1 mM PMSF, and 1× protease inhibitor cocktail. Briefly, 3 × 107 cells were used for nuclei isolation. Cells were lysed with Buffer A  and incubated on ice for 10 min. The lysate was layered onto Buffer B . Nuclei were pelleted at 10,000×g for 20 min and gently resuspended in Buffer C . CaCl2 was added to 3 mM, 8.5 units of MNase (Thermo Fisher) was added and the mixture was incubated at 37 °C for 10 min. The reaction was stopped by adding EGTA to 10 mM and incubated on ice for 5 min. Nuclei were collected by centrifugation at 10,000×g for 7 min and supernatant was collected as S1. The pellet was gently resuspended in Buffer D  and incubated for 2 h with head-to-head rotation at 4 °C. Nuclei were spun down at 10,000×g for 7 min and supernatant was collected as S2. S1 and S2 were pooled and further cleared at maximum speed for 5 min. Chromatin concentration was quantified spectroscopically (absorbance A260). For each immunoprecipitation, 100 μg chromatin was diluted with Buffer E  to 1 ml, and 1% from each sample was taken as input. For each reaction, 3 μg antibody listed in Supplementary Table −1 RNase A for 1 h at 37 °C in Tris-EDTA buffer. Samples were incubated in 0.5% SDS and 500 μg ml−1 Proteinase K with constant mixing at 56 °C overnight. Supernatant was collected from the beads. Input and nChIP DNA were purified with MinElute PCR purification kit (Qiagen) and analyzed with Agilent high sensitivity DNA chip using Agilent Technologies 2100 Bioanalyzer. Sequencing libraries were constructed from DNA samples including input and nChIP DNA with the Illumina TruSeq V3 library construction protocol (Illumina). Sequencing runs were performed on an Illumina NextSeq 500 in 75-base-pair (bp) single-end mode at the CCR-Sequencing Facility, National Cancer Institute, Frederick. RTA 1.8.70.0 software was used for basecall analysis. Reads were trimmed using Cutadapt v.1.18 to remove adapters and low-quality flanking regions. These reads were aligned to the mouse genome (MM10) using Bowtie2 2.2.6. with a mismatch error rates ≤0.05%. Reads that mapped to multiple locations in the genome were discarded.For nChIP60 using the following workflow:Genome browser views illustrate macroH2A and H2B enrichment for individual nChIP-seq samples expressed as read depth normalized to individual input controls, for example, KO1 represents macroH2A1 nChIP-seq sample of KO1 MEFs normalized to input control of KO1 MEFs. Supplementary Table macs2 callpeak -n $[sample]–bdg–nomodel–extsize 150 -t $[sample].input -c $[sample]macs2 bdgcmp -t $[sample]_treat_pileup.bdg -c $[sample]_control_lambda.bdg -m qpois -o $[sample]_qvalue.bdgbg2wig $[sample]_treat_pileup.bdg $[sample].wig mm10.chrom.sizeswigToBigWig -clip [sample].wig mm10.chrom.sizes [sample].bw84.The bigwigs (*.bw files) were viewed using the UCSC browser85. To tabulate macroH2A enrichment or LSH enrichment in 5 Kb tiles across the genome, wig files were converted to 5 Kb bins using a custom program. Resulting bins were the sum of all read coverage in the 5-Kb regions. To determine genome wide changes of macroH2A deposition in KO MEFs compared to controls, we compared macroH2A enrichment of six KO samples to macroH2A enrichment of six WT samples for individual 5Kb bins across the genome (for autosomes). Each bin was interrogated for statistically significant differences of KO/WT ratios using a two-tailed t-test. The percentage of differentially enriched bins is presented in Fig. 63. Peaks were selected with an FDR < 0.01, length >10 Kb, and log2 (Fold Change) >0.8. To compute macroH2A enrichment at protein coding genes, coverage of macroH2A enrichment at individual MM10 gene locations was extracted for each sample wig file using bigWigSummary84. Changes of macroH2A enrichment were computed for the gene body of protein coding genes  using the z-test for statistical analysis. The differences are computed by subtracting the mean value of macroH2A enrichment for Lsh knockout samples  for each gene from the mean value of corresponding wild-type samples . Upregulated genes  and downregulated genes (n = 241) have been previously identified by RNA-seq analysis conducted in Lsh−/− and Lsh+/+ MEF samples30. Peaks were called using Macs v. 2.2.660. For example, macs2 callpeak -n $[sample]–bdg–nomodel–extsize 150 -t $[sample].input -c $[sample]The outputs from call peak and bdgcomp created the pileup bedgraph (bdg) which was converted to a wig file using the custom bg2wig program. The pileups represent sequencing depth and background (input) normalized relative enrichment signal. Wigs were converted to bigwigs using the wigTotBigWig 4 program from UCSChttp://www.repeatmasker.org/). We aligned rodrep_ref.txt against MM10 genome using the Blat program from UCSC toolkit v. 36786. Best results were filtered using the following code:Peak overlap with repeat regions was computed using the rodent repeat database rodrep_ref.txt file from RepeatMasker version 4.1.0  that had been previously identified in embryonic stem cells61.Each repeats type was intersected with peaks using Bedops2 (RPKM + 1). MacroH2A and LSH occupancy levels (divided in fixed 5 Kb tiles) were transformed as the value of log2 (sum of reads + 0.1) to calculate Pearson correlation coefficient (Fig. P < 0.05 was considered a statistically significant difference.All ChIP-qPCR and RT-qPCR experiments were repeated at least three times as independent biological replicates and results are presented as mean ± SD. ChIP-seq samples as well as their respective input samples were derived as biologic replicates from three murine embryonic fibroblast cell lines for each genotype. Each cell line was derived from an individual embryo. All immunoblots were repeated at least three times with different biological samples and led to similar results. Micrographs are representative of a minimum of six images taken from at least three biological replicates. For Figs. ent Fig. , and to ent Fig. . StatistFurther information on research design is available in the Supplementary informationPeer Review FileReporting Summary


Inhibitors targeting the general RNA polymerase II (RNAPII) transcription machinery are candidate therapeutics in cancer and other complex diseases. Here, we review the molecular targets and mechanisms of action of these compounds, framing them within the steps of RNAPII transcription. We discuss the effects of transcription inhibitors in vitro and in cellular models (with an emphasis on cancer), as well as their efficacy in preclinical and clinical studies. We also discuss the rationale for inhibiting broadly acting transcriptional regulators or RNAPII itself in complex diseases. Changes in the regulation of gene transcription by RNA polymerase II (RNAPII) underlie virtually every complex human disease. Such changes have been most extensively documented in cancer, and gene expression signatures unique to particular forms of cancer are rich resources for identifying routes to novel, targeted therapies . MoreoveHere we review therapeutic strategies that target general RNAPII transcription regulators. We focus primarily on inhibitors of transcription-associated cyclin-dependent kinases (CDKs), found to be important targets of inhibitors that were originally sought for their effects on cell cycle progression. We also highlight non-CDK targets within the general transcriptional machinery, including RNAPII itself. For each target we discuss its mechanism of action during transcription and evidence for clinical efficacy of its cognate inhibitor, with a view toward understanding how balance is struck between therapeutic benefit and toxicity.RNAPII transcription occurs in three steps: initiation, elongation, and termination. Initiation involves the recognition of core promoter elements on DNA by RNAPII and general transcription factors and synthesis of the first few nucleotides of the RNA transcript. The steps in initiation were defined biochemically using purified factors and order-of-addition experiments, but larger assemblies of multiple factors may in fact engage promoters in vivo . A key sBefore RNA synthesis commences, RNAPII is also bound by Mediator, a 23-subunit assembly that further stabilizes the initiating RNAPII complex and acts as a physical link to gene-specific transcriptional activators bound to regulatory sites on DNA. Mediator is a dynamic complex composed of head, middle, tail, and kinase modules . The heaThe kinase module does not directly interact with activators, but instead seems to regulate a dynamic transition between Mediator bound at enhancer elements , and Mediator bound at the core promoter  Figure A. The kiFormation of the Mediator-RNAPII complex requires an interaction between the Mediator “head” module and the C-terminal domain (CTD) of RPB1, the largest RNAPII subunit . The CTDUnwinding of promoter DNA and initial RNA synthesis requires the engagement of the TFIIE and TFIIH complexes. TFIIH is a 10-subunit complex that includes ATP-dependent helicase subunits (XPB and XPD) and a kinase module composed of another transcription-associated CDK family member CDK7, its cognate cyclin CCNH, and MAT1 . PromoteUpon initiation, CDK7 phosphorylates the RNAPII CTD at the serine 5 position of the heptad repeat. This serves a dual purpose—it decreases the affinity of Mediator for RNAPII, resulting in its removal from the transcription complex, and it creates a binding site for enzymes involved in the addition of the methyl-guanosine cap structure to the 5′ end of the nascent mRNA transcript emerging from the RNAPII active site ,19 FiguA. CDK7 aAt many gene sites, elongation of RNAPII becomes paused ~50 nucleotides downstream of the transcription start site. Pausing allows for rapid and synchronous activation of transcription upon pause release, a regulatory feature shown to be critical for certain classes of genes such as those involved in stress responses or in embryonic development ,24. PausRecent studies have highlighted the importance of liquid–liquid phase separation (LLPS) in mediating some of the key events in the RNAPII transcription process. LLPS typically involves heterogeneous interactions between structurally disordered protein domains, forming large assemblies that behave as distinct compartments or “phases” . The RPBIn vivo, P-TEFb promotes phosphorylation of the RNAPII CTD at the serine 2 position in the heptad repeat. Serine 2 phosphorylation is a conserved mark of elongating RNAPII, is enriched near the 3′ ends of gene coding regions, and serves as a binding site for mRNA termination factors . CDK9 phThe previous section highlights CDKs as enzymes with multiple general roles in RNAPII regulation. The catalytic mechanism for RNA chain elongation by RNAPII is also a potential therapeutic target. X-ray crystallography studies have provided deep insight into these steps, which appear to be fundamental to DNA-dependent RNA polymerization reactions in all three branches of life. The RNAPII active site is comprised of structural elements formed by the large RNAPII subunits Rpb1 and Rpb2 . Catalys2+ ion binds the 3′ end of the RNA and a second Mg2+ ion binds the NTP pyrimidine scaffold . Hit comfunction . Dinacicfunction . A recenfunction . Other c50 of 3.2 nM, and the unique mechanism results in remarkable selectivity, as the IC50s for other CDKs are in the micromolar range [50 is almost 10-fold higher compared to CDK7. Preclinical studies have demonstrated the effectiveness of THZ1 to prevent cell proliferation and induce apoptosis in a variety of cancer types, such as high grade gliomas [THZ1 was identified through cell-based and kinase selectivity profiling of a library of inhibitors targeting the ATP-binding site of CDK7 . THZ1 isar range . CDK12,  gliomas , T-cell  gliomas , MYCN-de gliomas , small-c gliomas , triple  gliomas ,97, peri gliomas , and eso gliomas . Importa gliomas . Transit gliomas . Optimiz gliomas . A phaseTHZ1 treatment led to a dose-dependent decrease in RNAPII CTD phosphorylation at Ser2, Ser5, and Ser7 sites in multiple cancer cell lines ,96,98,99Owing to the fact that THZ1 specifically targets RNAPII transcription, it has been a useful tool for understanding the importance of general transcription mechanisms for sustaining cancer cell proliferation. Sensitivity to THZ1 in cancer cell lines correlates with the extent to which oncogenic, gene-specific transcription factors are overexpressed  ,95,99. A50 of 21 nM, demonstrating 25-fold greater potency than roscovitine (IC50 = 510 nM). BS-181 inhibited all other tested CDKs with IC50 values of 880 nM or greater [50 = 40 nM) was developed [BS-181 was derived by computational modelling, beginning with core fragments of roscovitine and subsequent docking studies. These studies identified a molecule favorable to the CDK7 active site over CDK2, CDK5, and CDK6 based on docking scores. In an in vitro kinase assay, BS-181 inhibited CDK7 with an IC greater . Further greater . Througheveloped , for whiCorticium simplex [50 = 1.8 nM) and reduced VEGF-induced migration of umbilical vein endothelial cells (HUVECs), without having an effect on phosphorylation of ERK1/2 and p38. Further insight into the mechanism of action for these compounds was gained via a high throughput screen with a panel of 402 kinases that revealed ROCKI and II, as well as the Mediator-associated kinases CDK8 and CDK19 [ds values of 17 and 10 nM, respectively, compared to >200 nM for ROCKI and ROCKII. Cortistatin A is selective for CDK8 and CDK19 due to remarkable shape complementarity with the ATP binding site. Crystallogaphy studies implicated a tryptophan residue in the ATP binding pocket unique to CDK8 and CDK19 in cation–π interactions with the dimethylamine group of cortistatin A [Cortistatin A is one of eleven natural steroidal alkaloids in the cortistatin family that have been isolated from  simplex ,104. Thend CDK19 . Quantitstatin A .-1 of cortistatin A led to a 71% decrease in tumor volume in a SET-2 acute myeloid leukemia (AML) xenograft mouse model. Surprisingly, suppression of AML growth was associated with increased expression of super-enhancer-linked genes. The mechanism for this repressive effect of CDK8/19 seems to involve phosphorylation of the transcription factor STAT1, which is prevented by cortistatin A [Both in vitro and in vivo mouse models of acute myeloid leukemia were used to demonstrate the antiproliferative activity of cortistatin A ,106. Forstatin A . These s50 in the 5–10 nM range). The co-crystal structure of CCT251545 bound to CDK8/cyclin C revealed that a loop region in the C-terminal domain of CDK8, far-removed from the kinase domain itself, folds over the active site and forms a hydrogen bond with the inhibitor. This unique binding mode likely contributes to the CDK8 specificity of CCT251545 [Links between Mediator kinase activity and STAT1 function in cancer have been strengthened by the study of two other inhibitors, CCT251545  and SEL1CT251545 . This loCT251545 . Gene exCT251545 . HoweverCT251545 .Whereas recently developed inhibitors of CDK7 and Mediator kinases derive their selectivity from amino acid residues unique to these kinases, selective CDK9 inhibitors recognize subtle structural features of the conserved ATP-binding pocket. As such, these inhibitors tend to retain significant affinity for other kinases, a likely explanation for their limited utility in preclinical and clinical studies . X-ray c50 of 6 nM for CDK9 and >470 nM for CDK1, CDK2, CDK3, CDK5, CDK6, and CDK7. In vivo studies with mice xenograft models of human acute myeloid leukemia demonstrated suitable pharmacokinetics and efficacy in reducing tumor growth. In primary adult T-cell leukemia and lymphoma, BAY 1143572 decreased RNAPII phosphorylation and expression of Mcl-1 and Myc [Although these and more recently developed CDK9 inhibitors have potent effects on cancer cells in vitro, there is as yet no data on their in vivo efficacy ,112. A p50 = 158 and 69 nM, respectively) over CDK7 and CDK9 . THZ531 exhibited dose- and time-dependent effects on proliferation and apoptosis in Jurkat T cell acute lymphoblastic leukemia cells. RNA-seq and ChIP-seq for RNAPII phosphorylated at serine 2 revealed that at low doses of THZ531, a small subset of genes involved in the DNA damage response displayed reduced expression. At higher doses sufficient to induce apoptosis, there was a global reduction in serine 2 phosphorylation at gene 3′ ends, reduced RNAPII processivity, and a global reduction in gene expression. The most highly affected genes encoded transcription factors associated with super enhancers and drivers of T-ALL growth, such as RUNX1, TAL1, and GATA3 [CDK12 and CDK13 crystal structures revealed a surface-exposed cysteine residue at a location corresponding to that of the THZ1 target cysteine in CDK7. Given the known roles of CDK12 in cancer, THZ1 was used as a starting point to develop THZ531, a selective CDK12/13 inhibitor [nd GATA3 .Tripterygium wilfordii Hook F, a medicinal plant that is also known as thunder god vine and that has been used in traditional Chinese medicine for centuries [Triptolide is a natural product that is purified from enturies . It is aenturies . In vitrenturies .Triptolide and some of its variants have been tested in clinical trials but have had limited success, primarily owing to their high toxicity . A recent attempt to circumvent this problem involved synthesis of glucose-conjugated triptolide . This coDrugs that target RNAPII itself have also been shown to be of therapeutic benefit in certain types of cancers. Actinomycin D (ActD) is commonly used as an experimental tool to inhibit transcription. It was originally developed as an antibiotic and was the first drug of this class to be shown to have therapeutic benefit in cancer. ActD is still used clinically in the treatment of Wilm’s tumor, rhabdomyosarcoma, and Ewing’s sarcoma . ActD isAlpha-amanitin (α-AMA) is a fungal toxin that selectively inhibits RNAPII-mediated transcription. It is known that α-AMA directly interferes with the mobile elements in the RNAPII catalytic center, the bridge helix, and the trigger loop, and is thought to prevent RNAPII translocation after nucleotide addition ,120. Co-Systemic administration of α-AMA is, unsurprisingly, extremely toxic, but genomic and proteomic studies have indicated patient populations where targeted use of α-AMA or its use as part of combination therapy could be rationalized. One study focused on drug-tolerant cells (DTCs) isolated from cancer cell lines treated with standard chemotherapeutic agents  or targeted agents  . The emePOLR2A gene encoding RPB1 [POLR2A is in close proximity to TP53 (encoding the tumor suppressor p53) on chromosome 17, and genomic deletions that remove TP53 in colorectal tumors frequently remove neighbouring genes, including POLR2A. Hemizygous loss of both TP53 and POLR2A was observed in some colorectal cancer (CRC) cell lines, which correlated with a reduced expression of RPB1 and a markedly increased sensitivity to α-AMA in vitro. These effects were demonstrated to be specifically due to the loss of POLR2A using engineered CRC cell lines. The α-AMA treatment sensitized these cells to treatment with standard chemotherapy agents used in CRC treatment, such as 5-fluorouracil, oxaliplatin, and 7-ethyl-10-hydroxy-camptothecin. These findings also translated into a potential therapeutic strategy in animal models. To avoid the toxicity associated with systemic α-AMA administration, α-AMA was covalently coupled to a monoclonal antibody against EpCam, an antigen overexpressed in adenocarcinoma. Mice xenografted with cell lines that were POLR2A+/- developed colorectal tumors, however tumor growth was significantly suppressed by i.p. injection of antibody-coupled α-AMA. A similar effect could be achieved by administration of POLR2A-directed siRNAs packaged in nanoliposomes [TP53 is a feature of many cancers, targeted RNAPII inhibition may be a generally applicable therapeutic approach in cancer.A second study revealed that many cancers might be vulnerable to treatment with RNAPII inhibitors due to hemizygous loss of the ing RPB1 . POLR2A iposomes . These tPOLR2A have been identified in a subset of benign meningiomas, common intracranial tumors that arise from the membranes surrounding the brain and spinal cord [POLR2A mutant tumors are phenotypically distinct from other characterized meningiomas and lack any of the known driver mutations associated with these tumors . This suggests that the mutations affect cell growth and division through a unique mechanism. The mutations cluster in the highly conserved “dock” domain of RNAPII, which interacts with TFIIB and is necessary for positioning of RNAPII with respect to TFIID and the correct transcription initiation site downstream of the core promoter. Interestingly, there was no evidence for aberrant mRNA start sites in transcriptome profiling experiments, and the mutations were instead found to misregulate specific genes involved in meningeal cell differentiation. More detailed study of transcription and mRNA processing at individual gene sites is needed to understand the mechanistic basis for these effects, however they further suggest that it is possible to identify subsets of individuals that may benefit from targeted manipulation of RNAPII activity.Somatic missense mutations in nal cord . POLR2A Targeting general transcriptional mechanisms in disease is a novel therapeutic approach that still requires validation in clinical settings. Nonetheless, initial indications are promising, and further tests of these agents in combination with other targeted or traditional therapies is likely to expand the potential clinical utility. Here, we have focused primarily on studies in cancers, however comparable states of transcriptional addiction are present in other disease states . As more


CEBPA gene are present in 10–15% of acute myeloid leukemia (AML) patients. The most frequent type of mutations leads to the expression of an N-terminally truncated variant of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), termed p30. While initial reports proposed that p30 represents a dominant-negative version of the wild-type C/EBPα protein, other studies show that p30 retains the capacity to actively regulate gene expression. Recent global transcriptomic and epigenomic analyses have advanced the understanding of the distinct roles of the p30 isoform in leukemogenesis. This review outlines direct and indirect effects of the C/EBPα p30 variant on oncogenic transformation of hematopoietic progenitor cells and discusses how studies of N-terminal CEBPA mutations in AML can be extrapolated to identify novel gain-of-function features in oncoproteins that arise from recurrent truncating mutations in transcription factors.Mutations in the  The intron-less CEBPA gene is located on chromosome 19.q13.1, and the usage of two translation initiation sites in the CEBPA mRNA can result in the expression of the full-length C/EBPα protein, p42 (42 kDa) and a shorter p30 isoform (30 kDa). The p30 isoform lacks two of three trans-activation elements (TE), but both isoforms share a third TE and the C-terminal basic-region leucine-zipper (bZIP) that is required for dimerization and DNA-binding.,6 Both C/EBPα p30 and p42 can homo- or hetero-dimerize with other C/EBP family members and regulate the expression of genes that govern cell differentiation, growth, survival, metabolism, and inflammation. The p42/p30 ratio can influence cell fate: high p42 expression blocks cell proliferation and results in differentiation, whereas increased p30 levels are associated with an immature cell state and inhibition of terminal differentiation into adipocytes and neutrophil granulocytes.,8,9 The expression ratio of the C/EBPα p42 versus p30 isoforms can be regulated by extracellular stimuli. Abundant nutrients and growth factors stimulate proliferation by enhancing the expression of an upstream open reading frame (uORF) in the CEBPA mRNA. Efficient uORF translation in nutrient-rich conditions promotes re-initiation of translation from the downstream start codon within the CEBPA-coding region, resulting in higher levels of C/EBPα p30.,9The transcription factor CCAAT/enhancer-binding protein alpha  and the SWI/SNF chromatin remodeling complex is important for its gene regulatory functions, highlighting the critical role of C/EBPα within complex networks of proteins that are involved in epigenetic control of gene expression.–13 In addition, C/EBPα p42 can directly interact with and stabilize p21 to inhibit cyclin dependent kinases, such as CDK4, thereby restraining cell cycle progression. Furthermore, direct suppression of E2F activity by interaction with p42 results in indirect inhibition of c-MYC expression and growth arrest.,14–18C/EBPα, other transcription factors play important roles in the development and differentiation of hematopoietic cells, including RUNX1, PU.1, and GATA2. De-regulation of the transcription factor network and changes in protein levels can affect cell fate decisions and promote leukemogenesis. Indeed, mutated or de-regulated master transcription factors are regularly identified as oncogenic drivers in leukemia.–27 Mutations in the CEBPA gene are found in 10–15% of de novo acute myeloid leukemia (AML) cases, predominantly in patients with normal karyotype acute myeloid leukemia (NK-AML).–30 The mutations are not equally distributed across the CEBPA locus but cluster in two hotspots in different regions of the gene  classification of myeloid neoplasms and acute leukemia, as it harbors few additional mutations, features a unique gene expression and immunophenotypic profile, and is associated with good prognosis.,31–36 The p30 variant is often the only functional C/EBPα protein in leukemia cells with biallelic CEBPA mutations, but the molecular mechanisms underlying oncogenic transformation by N-terminal CEBPA mutations is unclear.,28 In the following sections, we discuss recent advances in our understanding of C/EBPα p30-specific functions that might promote leukemogenesis.Besides C/EBPthe gene . N-termi22.1α p30 possesses functions that are distinct from p42 already emerged over a decade ago, when p30 was found to have the ability to bind to gene promoters and repress gene expression independently of p42 in hepatoma cells. Since then, several groups have provided evidence that p30 is a functional C/EBPα variant that is capable of selectively altering gene expression programs. For instance, p30 was shown to up-regulate the expression of Ubc9, which leads to a decrease in the p42/p30 ratio via addition of small ubiquitin-like modifier proteins (sumoylation) and subsequent down-regulation of p42 in leukemia cells. The resulting block in myeloid differentiation could be reversed by siRNA-mediated knockdown of Ubc9 and restoration of p42 levels. Furthermore, the peptidyl-prolyl isomerase PIN1 was up-regulated upon p30 overexpression. PIN1 is highly expressed in many cancers including AML and is known to increase the stability of the proto-oncogene c-Jun, resulting in a differentiation block. C/EBPα p30 was also shown to bind to the promoter of miR-181a and directly up-regulate its expression, which sensitizes AML cells to chemotherapy. Along the same lines, Hughes et al. discovered opposing roles of the p42 versus p30 C/EBPα isoforms in the regulation of the long non-coding RNA UCA1. While both isoforms were capable of directly binding to the promoter of UCA1, only p30 was able to induce UCA1-expression. In turn, high levels of UCA1 promote cell growth and proliferation by inhibiting the expression of the cell cycle regulator p27kip.The hypothesis that C/EBP2.2CEBPA-mutated AML and from samples of AML patients with biallelic CEBPA mutations. By comparing these C/EBPα-mutated leukemic cells with normal GMPs (granulocyte-macrophage progenitors), which represent their closest healthy counterparts, 102 genes were identified whose expression is de-regulated in human and mouse cells in response to C/EBPα mutations. Therefore, this gene set likely represents conserved effectors that might be functionally implicated in CEBPA-mutated AML. Only a small subset (20 genes) was up-regulated in C/EBPα-mutated cells, including ARPP21, NT5E, and ITGAX. Of these, CD73, the gene product of the NT5E gene, was also present at higher protein levels in cells with C/EBPα mutations. Two upstream enhancer regions were found to physically interact with the Nt5e promoter and strongly activate transcription, which was elegantly demonstrated by quantative analysis of chromosome conformation capture assays (3C-qPCR). Indeed, the C/EBPα p30 isoform was bound to an upstream enhancer of Nt5e and regulates CD73 expression levels. CD73 catalyzes the conversion of adenosine monophosphate to adenosine. As a result of up-regulated CD73 expression, elevated levels of adenosine stimulated proliferation and inhibited apoptosis in leukemia cells with C/EBPα mutations. In line with this, down-regulation of CD73 expression via clustered regularly interspaced short palindromic repeats interference (CRISPRi) (targeting the p30-bound enhancer) or RNA-interference significantly extended survival in a mouse model of CEBPA-mutated AML. Adenosine signaling requires members of the adenosine G-protein-coupled receptor family, which comprises four members: the A1 receptor, A2A receptor (A2AR), A2B receptor, and A3 receptor. Of these, only A2AR (encoded by the ADORA2A gene) was highly expressed in mouse and human C/EBPα-mutated cells. A2AR affects immune processes including pro-inflammatory cytokine secretion, macrophage-mediated phagocytosis, and C2 activation. In the context of CEBPA-mutated AML, adenosine signaling appears to function in a paracrine or autocrine manner through A2AR, as A2AR inhibition showed beneficial effects in a mouse model of CEBPA-mutated AML.While all the above-mentioned studies focused on individual genes, a recent study by Jakobsen et al. investigated global p30-dependent gene expression changes. The authors performed comparative analyses of transcriptomic data derived from a mouse model of 2.3α isoforms as well as epigenetic marks were assessed to characterize isoform-specific differences in p42- versus p30-expressing cells. Although both C/EBPα isoforms share the C-terminal DNA-binding domain, a subset of genomic regions  was bound exclusively by either the p42 or the p30 isoform. Interestingly, most of these regions  were occupied by the oncogenic p30 protein. While genomic regions with shared C/EBPα isoform binding as well as p42-specific binding sites harbored high-affinity C/EBPα consensus motifs, p30 localized to low-affinity C/EBPα binding sites. In addition, p30-occupied regions were enriched for transcription factor binding motifs for ERG, FLI1, and PU.1 proteins. In contrast, co-binding with the transcription factor HLF appears to be exclusive for p42, as only p42 binding sites harbored HLF consensus motifs and HLF expression was significantly down-regulated in p30-expressing cells. The findings of differential distribution of transcription factor binding motifs at isoform-exclusive genomic sites suggest that p42 and p30 can interact with different co-factors on chromatin. In addition to different distribution of transcription factor binding motifs, p42-bound regions were associated with lower histone 3 lysine 27 (H3K27) acetylation and lower histone 3 lysine 4 (H3K4) mono-methylation, while p30-bound regions as well as loci occupied by both p42 and p30 displayed high levels of these histone marks. As both H3K27 acetylation and H3K4 monomethylation are characteristics for enhancer regions, these results suggest that p30 preferably binds to primed enhancer regions, while p42 might be less dependent on the prior presence of chromatin marks that are associated with active transcription. This is consistent with data showing that C/EBPα can act as a pioneer factor in cooperation with other transcription factors, but it is not known if this feature is preserved in the truncated p30 variant of the protein.,10 Based on these observations, it is proposed that higher levels of p30 protein as found in patients with CEBPA mutations enable p30 binding to low-affinity C/EBPα motifs at active and/or primed enhancers. In general, chromatin binding of either C/EBPα isoform was positively correlated with transcriptional activation, although the transcriptional activity of p30 is weaker than p42. This is in line with the observation that genes that are regulated by common p42/p30 binding to regulatory regions were more frequently down-regulated when p42 was missing. However, the expression of a different group of genes that is regulated by common p42/p30 binding was up-regulated in the absence of p42. Another recent publication also demonstrates opposing effects of C/EBPα isoforms on gene regulation. Chromatin binding of the transcription factor MYB, which is often de-regulated in leukemia at p30-specific sites was correlated with activated transcription, while the expression of genes in the proximity of regions bound by MYB together with p42 was predominantly repressed. These data point toward specific generegulatory functions of p30 that result in aberrant gene expression and oncogenic transformation.In the same study, global chromatin association patterns of C/EBP2.4α p30 variant can also exert specific functions through its interaction with epigenetic regulators. Affinity purification followed by mass spectrometry and co-immunoprecipitation studies found that the p30 isoform preferentially interacted with the MLL1 methyltransferase complex, which catalyzes H3K4 trimethylation.,47 Consistently, there was a large overlap in global chromatin association of C/EBPα p30 and the MLL1 complex. These data indicate a cooperation of p30 with the MLL1 methyltransferase complex in the regulation of genes that are critical for the oncogenic effect of p30. CRISPR/Cas9-mediated mutagenesis revealed that p30-expressing cells are dependent on a functional MLL1 protein. Disruption of the MLL1 complex function by small-molecule-mediated inhibition of the MLL1-Menin or MLL1-WDR5 interaction resulted in impaired proliferation, induction of differentiation, and elevated levels of apoptosis of CEBPA-mutated cells.,47The C/EBPα p30 protein represents a functional isoform of C/EBPα that can exert oncogenic effects through binding to enhancers and promoters and/or via preferential interaction with epigenetic modifiers and co-factors, which results in the specific regulation of p30-target genes.Taken together, these data demonstrate that the C/EBP33.1CEBPA mutations in AML, other scenarios exist where a truncated transcription factor isoform retains the capacity of actively changing gene expression in cancer. Patients with Down syndrome have a high risk of developing a transient myeloproliferative disorder, which frequently develops into acute megakaryocytic leukemia. In this context, mutations in the transcription factor GATA1 result in the expression of an N-terminally truncated protein that was termed GATA1s. Like p30, GATA1s retains the ability to bind DNA and can act as a transcriptional activator. Knockdown of GATA1 in a GATA1s-only expressing cell line resulted in de-regulation of genes associated with proliferation, differentiation, and cell death, some of which could be direct GATA1s targets. For instance, GATA1s can bind to GATA1-binding sites in the promoter region of the IL1A gene and regulate its protein levels. Additionally, the selective binding of GATA1s, but not GATA1, to regions corresponding to 140 genes implicated in diverse cellular pathways was recently demonstrated. Similar to the C/EBPα isoforms, the genomic regions that were bound by the truncated GATA1s isoform were associated with distinct transcription factor binding motifs, suggesting altered, isoform-specific interactions. Furthermore, the presence of GATA1s in erythroid cells was associated with dysregulated H3K27 methylation and altered chromatin accessibility. Thus, GATA1s could contribute to cell growth and survival by changing the epigenetic landscape to drive the expression of a subset of genes via differential co-factors.Beyond N-terminal 3.2 In contrast, RUNX1 levels are increased in many cancers, especially in AML. Interestingly, both up- and down-regulation of the transcription factor GATA2 can promote leukemogenesis.–58 These findings exemplify the strong impact that transcription factor imbalance can have on proliferation, differentiation, and death of hematopoietic progenitor cells. Furthermore, they highlight the need to elucidate the molecular mechanisms by which mutated or de-regulated transcription factors contribute to leukemogenesis.De-regulated expression of transcription factors is recurrently observed in leukemia, highlighting the critical importance of balanced transcription factor expression for hematopoietic homeostasis. For instance, deletion of an upstream regulatory element causes down-regulation of PU.1 expression and leads to AML development.3.3α p30 isoform can interfere with normal transcriptional regulation. The identification of genomic regions that are exclusively bound by p30 indicates that this N-terminally truncated variant of C/EBPα can participate in active transcriptional regulation of distinct gene sets. Thus, the recent novel insights into the oncogenic mechanisms of C/EBPα p30 illustrate how a truncating mutation in a transcription factor can result in a functional isoform that has gained novel molecular functions to enable AML development. High levels of p30 expression and/or the absence of p42, which is observed in CEBPA-mutated AML, might allow binding of p30 to genomic regions that harbor low-affinity C/EBPα motifs, thereby changing global C/EBPα-dependent gene expression. As p30 is the only functional C/EBPα isoform present in the majority of AML with biallelic CEBPA mutations, genes that are aberrantly regulated by p30 are likely to include important oncogenic effectors. However, the p30-mediated activation of genes is only one of several aspects by which isoform imbalance in CEBPA-mutated AML could promote leukemogenesis. Loss of p42 leads to de-repression of genes as well as down-regulation of genes that are activated by p42. As the p30 isoform lacks two of three TEs, p30 exhibits reduced capacity to activate gene expression. The p30-induced up-regulation of specific gene sets could therefore result from altered patterns of co-factor binding and/or p30-specific interaction with other transcription factors, a concept that is supported by the finding that p42- versus p30-bound genomic regions exhibit different repertoires of transcription factor binding motifs. While HLF could be a p42-specific co-factor, factors such as ETS, ERG, and FLI1 show higher association with p30. In contrast, the known C/EBPα co-factor MYB might not distinguish between C/EBPα isoforms for chromatin binding, but exert differential gene regulatory effects depending on its association with either C/EBPα isoform. These results highlight the strong context-dependent influence of co-factor interactions on protein function. C/EBPα-specific co-factors can also be epigenetic regulators, such as the MLL1 histone methyltransferase complex. Thus, it is possible that the interaction of p30 with the epigenetic machinery is important for activation of transcription in cooperation with other transcription factors.Several recent studies have proposed mechanisms by which the oncogenic C/EBP4α is a gain-of-function allele that mediates oncogenic transformation via de-regulation of transcriptional programs. C/EBPα p30 displays altered chromatin binding properties that might depend on its interaction with other transcription factors and on different chromatin states. Specific interaction partners of p30 could promote binding of p30 to novel genomic regions to activate gene expression. The resulting global epigenomic and transcriptional changes that lead to oncogenic transformation demonstrate how aberrant expression of truncated yet functional isoforms of transcription factors can contribute to leukemogenesis (Taken together, recent evidence is compatible with a scenario where the truncated p30 isoform of C/EBPogenesis .


The majority of eukaryotic genes produce multiple mRNA isoforms by using alternative poly(A) sites in a process called alternative polyadenylation (APA). APA is a dynamic process that is highly regulated in development and in response to extrinsic or intrinsic stimuli. Mis-regulation of APA has been linked to a wide variety of diseases, including cancer, neurological and immunological disorders. Since the first example of APA was described 40 years ago, the regulatory mechanisms of APA have been actively investigated. Conventionally, research in this area has focused primarily on the roles of regulatory cis-elements and trans-acting RNA-binding proteins. Recent studies, however, have revealed important functions for epigenetic mechanisms, including DNA and histone modifications and higher-order chromatin structures, in APA regulation. Here we will discuss these recent findings and their implications for our understanding of the crosstalk between epigenetics and mRNA 3'-end processing. Cleavagractions . A majorractions . Differeractions . As suchractions . APA is ractions . HoweverSimilar to other steps of gene expression, APA is highly developmental stage- and tissue-specific. As the vast majority of the cells in multi-cellular organisms contain an identical genome, these differences are most likely caused, directly or indirectly, by epigenetic changes. Epigenetic mechanisms refer to reversible and heritable alterations that modulate gene expression without changing the DNA sequence . In eukaAll of these epigenetic mechanisms are known to regulate transcription. For example, DNA methylation at promoters is known to repress transcription, in part by preventing transcription factors from binding to DNA . DNA metvia modulating transcription, we will begin by discussing the links between transcription and APA. The processes of transcription and mRNA 3'-end processing are tightly coupled. mRNA 3'-end processing factors are recruited to the transcription machinery as early as the pre-initiation complex and are believed to traverse the gene body with RNAPII  prevent usage of the intronic Herc3 PAS and (2) block expression of Nap1l5 on the maternal allele  are highly prevalent in eukaryotic genomes. They are typically found in clusters and the chromatin regions containing TREs are generally silenced through DNA methylation and repressive histone modifications such as H3K9 methylation . Interesf RNAPII . In the f RNAPII . MechaniMost studies related to epigenetics and APA have focused on regulation of APA by epigenetic mechanisms, but recent evidence highlights modulation of epigenetics by APA. In 2006, the Yamanaka group demonstrated that differentiated cells can be reprogrammed to a stem cell-like state by over-expressing four genes . The effvia the viral protein NS1 (Changes in mRNA 3'-end processing can also physically disrupt 3D genome organization. Influenza virus infection leads to host gene shut-off. One mechanism by which the virus inhibits host gene expression is by inhibiting host mRNA 3'-end processing tein NS1 . A recentein NS1 . Elongattein NS1 . Similartein NS1 . These sAPA continues to gain appreciation as a major strategy used by cells to fine-tune gene expression. Researchers are increasingly mapping APA patterns and studying its regulatory mechanisms across organisms, cell types, and during cell fate transitions. Recent advances in the field have clearly demonstrated that epigenetic mechanisms, including DNA and histone modifications and chromatin structures, play an important role in APA regulation. Mechanistically, many of these epigenetic factors regulate APA indirectly through modulating RNAPII elongation and pausing. For future studies, it will be critical to identify and characterize the factors that mediate the communication between DNA/chromatin and RNA processing. For splicing, a number of splicing regulators have been shown to bind to specific histone mark readers, thereby mediating the regulation of splicing by chromatin features. Such interactions are currently lacking for APA regulation. For example, in the AAE complex mentioned earlier, EDM2 recognizes histone marks and ASI1 most likely binds to RNA, thereby linking chromatin directly to RNA. Future studies will determine if similar complexes also exist in metazoans. Finally additional efforts are needed to understand the biological consequences of epigenetics-mediated APA regulation in development and in diseases.LS and YS contributed to the writing of this article. Both 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.


In this study, Kang et al. sought to understand the underlying principles that mediate transcriptional memory and reactivation in the daughter cells after mitosis. They used ChIP-seq on synchronized cells at different stages after mitosis to generate genome-wide maps of histone modifications, and combined with EU-RNA-seq and Hi-C analyses, they found that during prometaphase promoters, enhancers, and insulators retain H3K4me3 and H3K4me1, while losing H3K27ac, thus providing new insights into the histone modification landscape during cell division. During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the underlying principles that mediate transcriptional memory and reactivation in the daughter cells remain unclear. Here, we used ChIP-seq on synchronized cells at different stages after mitosis to generate genome-wide maps of histone modifications. Combined with EU-RNA-seq and Hi-C analyses, we found that during prometaphase, promoters, enhancers, and insulators retain H3K4me3 and H3K4me1, while losing H3K27ac. Enhancers globally retaining mitotic H3K4me1 or locally retaining mitotic H3K27ac are associated with cell type-specific genes and their transcription factors for rapid transcriptional activation. As cells exit mitosis, promoters regain H3K27ac, which correlates with transcriptional reactivation. Insulators also gain H3K27ac and CCCTC-binding factor (CTCF) in anaphase/telophase. This increase of H3K27ac in anaphase/telophase is required for posttranscriptional activation and may play a role in the establishment of topologically associating domains (TADs). Together, our results suggest that the genome is reorganized in a sequential order, in which histone methylations occur first in prometaphase, histone acetylation, and CTCF in anaphase/telophase, transcription in cytokinesis, and long-range chromatin interactions in early G1. We thus provide insights into the histone modification landscape that allows faithful reestablishment of the transcriptional program and TADs during cell division. Mitosis marks a dramatic transition during which cells move from a transcriptionally active to a largely repressed state in which most genes are transcriptionally inactive . This drcis-regulatory elements (cis-REs), such as promoters and enhancers, and regulate gene expression via interaction with transcription factors. H3K4me3 and H3K27ac are highly enriched at active promoters near the transcription start site (TSS) and positively correlated with transcription. Enhancers play a crucial role in the activation and fine-tuning of their target promoters. Enhancer elements can exist in two major chromatin states, primed or active. Primed enhancers (PEs) are marked by H3K4me1/2 and their target genes are weakly or not expressed, whereas active enhancers (AEs) are additionally marked by the histone acetyltransferases CBP/p300-mediated H3K27ac and are associated with actively transcribed genes , and DNA loops are lost during prometaphase and reestablished in G1 phase . TADs arIn this study, we combined histone modification ChIP-seq, EU-RNA-seq, and Hi-C from prometaphase to G1 phase to comprehensively examine on a genome-wide level the temporal relationship between histone modification binding, nascent transcription, and long-range chromatin interactions during the mitosis–G1 transition. We found that H3K4me3 remains associated with almost all promoters and that H3K4me1 is highly retained at enhancers of cell type-specific genes during prometaphase. In contrast, H3K27ac is largely reduced during prometaphase, although a subset of H3K27ac remains at enhancers of cell type-specific genes and promotes early expression of these cell type-specific genes. During the anaphase/telophase transition, most promoters gain H3K27ac, and this is essential for the accurate transmission of gene expression programs during cell division. Furthermore, TAD boundaries during prometaphase remain associated with H3K4me3 and H3K4me1 in the absence of CTCF, but CTCF is recruited and enriched at the boundaries during anaphase/telophase together with H3K27ac before the appearance of loops, TADs, and compartments. Altogether, the multilevel data of epigenetic landscape, nascent transcription, and TAD interaction shed light on not only the temporal order of genome organization after mitosis but also indicate a potential bookmarking role for histone modifications in accurate re-establishment of transcriptional activation pattern and long-range chromatin interactions.Supplemental Fig. S1A). Thus, our synchronization approach enables us to collect enriched populations of cells at prometaphase, anaphase/telophase, and interphase, respectively.Transcription is reactivated at the time of mitotic exit, as the NE starts reassembling and RNA Pol II globally rebinds to the chromatin . It is tSupplemental Fig. S1B,C). To comprehensively examine the extent and genomic localization of mitotic occupancy of histone modifications, we performed ChIP-seq for H3K4me3, H3K4me1, and H3K27ac in U2OS and RPE1 cell lines during interphase, prometaphase, and anaphase/telophase . When there is a global signal change under different experimental conditions , typical normalization by the total number of mapped reads can be misleading since similar distributions of positions are sampled during sequencing. It is thus often challenging to quantitatively compare histone modification ChIP-seq data sets from different phases of the cell cycle. To overcome this, we used a spike-in based normalization strategy, whereby adding an equal amount of exogenous chromatin from a different species into each sample can be used as a more accurate normalization control  or active enhancers . We performed immunofluorescence in asynchronous cells and Western blotting in histone extracts from interphase, prometaphase, and anaphase/telophase enriched cells. We observed that all three histone modifications  were detected in all phases of mitosis from both U2OS and RPE1 cells , PE , and AE  elements based on our ChIP-seq data  and H3K4me1 peaks were depleted at TSS . Only a small fraction of interphase AEs retained H3K27ac in prometaphase (18.0%). Of AEs, 69.2% lost H3K27ac, but retained H3K4me1 in prometaphase cells . We found that the majority of PEs remained stable in prometaphase and anaphase/telophase  similar to H3K4me3+ promoters . These data suggest that promoters and enhancers continue to retain H3K4me3 and H3K4me1 respectively, as potential bookmarks, although they lose H3K27ac during prometaphase.We next wanted to determine how gene regulatory elements “remember” their histone modification dynamics across mitosis. To answer this question, we selected all promoters and enhancers from interphase and compared the dynamics of each histone modification from interphase to prometaphase or to anaphase/telophase cells. Promoters can be marked by either H3K4me3 only or by both H3K27ac and H3K4me3. We found that the majority of H3K4me3+ promoters remained in prometaphase (98.3%). In contrast, only 1.1% of promoters remained associated with H3K27ac in prometaphase and 87% of H3K4me3+ promoters were able to regain H3K27ac in anaphase/telophase . Notably, promoters retaining H3K4me3 during prometaphase, were strongly enriched for genes involved in fundamental cellular processes, such as RNA and protein metabolism, whereas AEs either retaining or losing H3K27ac during prometaphase were predominantly linked to differentiation or development-related genes in both cell lines. Specifically, we observed that AEs losing H3K27ac, but retaining H3K4me1 were most strongly associated with genes involved in bone mineralization in U2OS . Together, these results indicate that enhancers use H3K4me1 to maintain their cellular identity upon loss of H3K27ac throughout mitosis.Next, we asked whether continuous retention of H3K4me1 during prometaphase associates with a bookmark of cell type-specific genes. For this, we used U2OS cells, which are derived from cells of mesenchymal origin that differentiate to osteoblasts. There are several genes identified as essential for bone formation and differentiation to osteoblasts. These so-called osteoblast-specific genes include and SOST . Strikincis-REs, we performed de novo motif analysis of the bookmarked elements in U2OS cells. Our analysis showed that both AEs and PEs were preferentially enriched with motifs of the activator protein-1 (AP-1) family of TFs including FOSL2, JUN-AP1, FRA2, FRA1, and JUNB . Then, we captured nascent RNA by adding biotin-azide and generated cDNA libraries for sequencing. As there was a large difference in the amount of nascent RNA between interphase and mitotic cells, we used external controls that appropriately normalize the RNA-seq data . Consistent with the previous study, we found a hierarchy of gene reactivation at the mitosis to G1 transition (Histone modifications are found on mitotic chromatin with either a stable (H3K4me1/3) or reduced (H3K27ac) abundance when compared with interphase chromatin . Togetheansition . We alsoansition B. Gene Oansition C. HoweveR = 0.60) . AEs are associated with active transcription by bringing DNA loops physically close to gene promoters. However, for AEs, either H3K27ac or H3K4me1 level in mitosis was not positively correlated with gene expression  D. FurtheSupplemental Fig. S3). We thus asked whether H3K4me1 occupancy throughout mitosis associates with rapid transcriptional activation of cell type-specific genes. We obtained the list of 113 genes specifically expressed in bone or bone marrow, which are also expressed in U2OS, using the Tissue-specific Gene Expression and Regulation (TiGER) database . We then washed out both nocodazole and A-485 and released cells for 1 h or 24 h. Reduced H3K27ac binding from prometaphase chromatin was able to regain as cells exit mitosis to enter 1-h cytokinesis or 24-h next cell cycle . Second, to inhibit H3K27ac regaining from anaphase/telophase, we released cells from mitotic arrest while treating with A-485 for 1 or 3 h. The H3K27ac levels successfully decreased in both 1-h and 3-h treatment with A-485 . Next, nascent transcripts were pulse-labeled with the EU followed by sequencing and normalized by spike-in controls . We confirmed by EU-RNA-seq analysis that the biological replicates for each treatment were highly correlated  and that A-485 treatment resulted in extensive transcriptional changes for all treatments .Mitotic occupancy of histone modifications is associated with postmitotic gene activation suggestive of mitotic bookmarking. However, a recent study showed that inhibition of mitotic BRD4 binding did not affect transcriptional activation, suggesting that mitotic occupancy can be passively bound to mitotic chromatin and dispensable for transcription . To test2 fold change  upon A-485 treatment. When inhibiting mitotic H3K27ac, 56% (48% down and 8% up) of the genes in prometaphase and 45% (29% down and 16% up) of the genes in cytokinesis were dysregulated. Since the length of the cell cycle is ∼24 h for dividing mammalian cells, we let cells enter the next cell cycle upon mitotic inhibition of H3K27ac. Surprisingly, 45% (22% down and 23% up) of the genes in 24-h release cells were dysregulated, suggesting that mitotic H3K27ac is required for maintaining transcriptional memory. H3K27ac begins to get regained from anaphase/telophase . We assessed the reproducibility of Hi-C data between two biological replicates and observed a strong Pearson correlation coefficient at all time points . In agreement with previous observations, Hi-C contact maps showed that chromosomal interactions were no longer observed in mitotic cells . In concordance with our ChIP-seq analysis, immunofluorescence and Western blot analyses showed that CTCF is largely dispersed to the cytoplasm in prometaphase, then recruited on chromatin in anaphase/telophase . We then analyzed the distribution of CTCF binding peaks at the TAD boundaries in each cell cycle phase. As previously suggested, interphase CTCF binding was enriched at the TAD boundaries. We did not observe any enrichment at the boundaries in prometaphase, possibly due to the limited numbers of binding sites during this stage. Strikingly, anaphase/telophase CTCF binding was strongly enriched at the boundaries even higher than that seen in interphase  and 79% of H3K27ac   (1 peaks) B interph1 peaks) E,F. Thus1 peaks) I, indica1 peaks) J, indica1 peaks) K,L. TogeHere, we provide a temporal order of genome organization during and after mitosis. During prometaphase, histone methylation binding is largely retained, however only low levels of transcription occur and no long-range chromatin interactions are detected. Because histone modifications are established first, we investigated whether mitotic histone modification binding contributes to gene reactivation or to the formation of long-range chromatin interactions. We observed that levels of H3K4me3 and H3K4me1 are stable throughout mitosis at promoters and enhancers, respectively, and both at insulators. H3K4me1 remains at enhancers of cell type-specific genes and TFs binding motifs, which regulate cell type-specific gene expression during prometaphase. This indicates that histone methylations act as a widespread epigenetic memory of regulatory information previously active in the mother cells. Limited H3K27ac remains associated with mitotic chromatin for the accurate transmission of transcription programs and early expression of cell type-specific genes. Loss of H3K27ac in prometaphase is recovered in anaphase/telophase preferentially at promoters and insulators. This recovery of H3K27ac by anaphase/telophase is required for gene reactivation and is particularly relocalized at the TAD boundaries. When cells enter cytokinesis, most of the genes are transcribed. Long-range chromatin interactions gradually increase from cytokinesis, and are complete between 90 and 120 min G1. In addition to H3K27ac, CTCF lost in prometaphase is recruited in anaphase/telophase at the TAD boundaries, which may be involved in the reformation of TADs in the daughter cells. Overall, the genome is orderly reformed in which histone methylations are retained throughout mitosis, followed by histone acetylation and CTCF in anaphase/telophase, transcription in cytokinesis, then long-range interactions in 90 min G1 .cis-REs. First, H3K4me3 bookmarks promoters of genes during prometaphase. Ninety-eight percent of promoters retain H3K4me3 during prometaphase . Second, H3K4me1 bookmarks enhancers of cell type-specific genes during prometaphase. Cell type-specific genes tend to contain H3K4me1 during prometaphase and to be expressed early in the mitosis–G1 transition  that acetylates histones H3 and H4 such as H3K14ac, H3K27ac, H3K122ac, and H4K16ac . It bindRecently, a genome-wide RNA pulse-labeling study showed that some genes are actively transcribed in prometaphase arrested cells, and described waves of gene reactivation during mitotic exit . Using tMultiple studies show that CTCF remains associated with mitotic chromosomes using imaging and Western blot analyses , which p2. Cells were synchronized using thymidine and nocodazole treatments. The mitotic cells were collected by mitotic shakeoff. A-485  was used to inhibit H3K27ac levels. See the Supplemental Material for detailed description.U2OS cells were cultured in DMEM (Gibco) and 10% FBS. RPE1 cells were cultured in DMEM-F12 (Gibco) and 10% FBS. Cells were incubated at 37°C and 5% COCells were grown on glass coverslips and fixed with 4% paraformaldehyde in PBS for 5 min at room temperature. Fixed cells were permeabilized with cold PBS containing 0.5% Triton X-100 for 5 min. Cells were blocked with 1% BSA in PBS for 1 h at room temperature and incubated in primary antibody in blocking buffer overnight at 4°C, followed by secondary antibody for 1 h at room temperature. Cells were then briefly stained with 1 μg/mL Hoechst 33342 (Molecular Probes H-1399) in PBS and mounted in VectaShield (Vector Laboratories). Images were acquired in a Zeiss LSM710 confocal microscope. Images were analyzed and prepared for presentation in Photoshop.g for 10 min at 4°C. The supernatant containing the cytoplasmic fraction was transferred and saved. The pellet was resuspended in lysis buffer  for 30 min on ice, followed by centrifugation at 12,000g for 20 min at 4°C. The supernatant was collected as the nuclear fraction.Cells were resuspended in hypotonic buffer  for 10 min on ice , followed by centrifugation at 500For histone extraction, the protocol is adapted from previous work . BrieflyThe following antibodies were purchased from the indicated commercial sources: anti-H3K4me3 (Abcam ab8580), anti-H3K4me1 (Abcam ab8895), anti-H3K27ac (Abcam ab4729), anti-CTCF (Active Motif 61311), anti-spike-in (Active Motif 61686), anti-α-Tubulin (Sigma T5168), anti-Histone H3 , and anti-H3S10p .6) were cross-linked with 1% formaldehyde for 10 min, and ChIP-seq was performed as described previously  were cross-linked with formaldehyde. Chromatin was digested with a restriction enzyme MboI (NEB), biotinylated with biotin-ATP (Life Technologies), and then ligated with T4 DNA ligase (NEB). DNA was purified and sheared with Covaris LE220 instrument (Covaris). Biotinylated DNA interactions were pulled down with Dynabeads MyOne Streptavin T1 Beads (Life Technologies) and sequenced in a NovaSeq 6000 sequencing system (Illumina). See the Supplemental Material for detailed information about data analysis.Hi-C was performed using the in situ method as previously described . BrieflyThe Gene Expression Omnibus accession number for the ChIP-seq, EU-RNA-seq, and Hi-C data reported in this study is GSE141139.