With the discovery that more than half of human cancers harbor mutations in chromatin proteins, deregulation of epigenetic mechanisms has been recognized a hallmark of malignant transformation. Post-translational modifications (PTMs) of histone proteins, as main components of epigenetic regulatory machinery, are also broadly accepted as therapeutic target. Current “epigenetic” therapies target predominantly writers, erasers and readers of histone acetylation and (to a lesser extent) methylation, leaving other types of PTMs largely unexplored. One of them is the phosphorylation of serine 10 on histone H3 (H3S10ph).H3S10ph is emerging as an important player in the initiation and propagation of cancer, as it facilitates cellular malignant transformation and participates in fundamental cellular functions. In normal cells this histone mark dictates the hierarchy of additional histone modifications involved in the formation of protein binding scaffolds, transcriptional regulation, blocking repressive epigenetic information and shielding gene regions from heterochromatin spreading. During cell division, this mark is essential for chromosome condensation and segregation. It is also involved in the function of specific DNA–RNA hybrids, called R-loops, which modulate transcription and facilitate chromosomal instability. Increase in H3S10ph is observed in numerous cancer types and its abundance has been associated with inferior prognosis. Many H3S10-kinases, including MSK1/2, PIM1, CDK8 and AURORA kinases, have been long considered targets in cancer therapy. However, since these proteins also participate in other critical processes, including signal transduction, apoptotic signaling, metabolic fitness and transcription, their chromatin functions are often neglected.H3S10ph and enzymes responsible for deposition of this histone modification are important for chromatin activity and oncogenesis. Epigenetic-drugs targeting this axis of modifications, potentially in combination with conventional or targeted therapy, provide a promising angle in search for knowledge-driven therapeutic strategies in oncology. Recent large-scale cancer genome projects like The Cancer Genome Atlas highlighted the role of epigenetic alterations in cancer development, as they revealed that about half of human cancers harbors mutations in chromatin-modifying proteins . Post-trPhosphorylation of serine 10 on histone H3 (H3S10ph) is an unique histone modification as it is involved in two structurally opposed processes: transcriptional activation and chromatin relaxation, or chromosome compaction during cell division –14. ThisIn this review, we address the role of H3S10 phosphorylation in chromatin biology. We present multiple H3S10 kinases that are known for their extra-chromatin functions; for example, in signaling cascades typically activated in cancer, we discuss their role in cancer and their inhibition as a potential therapeutic strategy.In contrast to other modifications of H3 tail like H3K9me2 or H3K9ac, H3S10ph mark does not become erased as cell enters mitosis , but itsS. cerevisiae, H3S10ph is required for recruitment of the histone deacetylase HST2, which mediates deacetylation of H4K16, freeing the H4 tail to interact with the surface of neighboring nucleosomes and promoting fiber condensation [In addition to displacement of HP1, H3S10ph can affect interactions of chromatin also with other proteins, regulating mitotic chromosome dynamics. For example, in ensation . Anotherensation . Consistensation , but is ensation . Moreoveensation . H3S10phensation .Interestingly, H3S10ph is not uniformly inherited through the cell cycle. It is predominantly associated with an “old” H3 at early mitosis and appears in “new” H3 with the progression of mitosis reaching symmetrical distribution . InhibitRecent studies reveal a new role of H3S10ph mark, linking its deposition and chromatin structure to genome integrity. H3S10ph mark is abundantly deposited within actively transcribed regions forming hybrid structures composed of a nascent RNA, DNA template and a displaced nontemplate single-stranded DNA, called R-loops , 44. R-lSince R-loops are a potential threat to genomic integrity, it is particularly interesting that their formation is associated with certain chromatin modifications, including H3S10 phosphorylation. H3S10ph occurs specifically at least at a subset of R-loop-forming regions. Overexpression of RNAse H, resolving R-loops, abrogates this effect, indicating that H3S10 phosphorylation is a specific cellular response to R-loops. H3S10ph at these R-loops leads to chromatin compaction, and dysregulation of H3S10 phosphorylation leads to increased genomic instability . HistoneThe deleterious role of H3S10 at the sites of DNA damage has been also suggested by the studies by Sharma . Ionizinc-FOS and c-JUN, resulted in their rapid transcriptional induction. Interestingly, the same regions were reported to be especially susceptible to lysine hyperacetylation, suggesting a link between H3 phosphorylation and acetylation. In fact, a more detailed analysis showed that histone H3 on c-FOS- and c-JUN-associated nucleosomes becomes doubly-modified, the same H3 tails becoming both phosphorylated and acetylated upon gene activation [Interphase chromatin is broadly occupied by H3S10ph, especially in gene-rich regions. H3S10 phosphorylation is abundant at domains of early DNA replication timing and correlates negatively with H3K9me2 and lamina-associated domains, suggesting that H3S10ph plays a role in activation of transcription . Indeed,tivation . Dual H3tivation , 55, 56.FOSL1 gene [In vitro pull-down assays with N-terminal tail of histone H3 phosphorylated at S10 followed by mass spectrometry identified 6 members of 14–3–3 protein family as H3S10ph binding partners , 58. 14-SL1 gene , 63. H3SSL1 gene . In addiSL1 gene .Lysine K9 in H3 lies in the immediate neighborhood to S10. K9 carries not only acetylation mark, recruiting factors associated with transcriptional activation, but also methylation mark, which binds factors associated with transcriptional repression . This suIt is important to mention that not all readers of methylated K9 are sensitive to H3S10 phosphorylation. Likewise, S10ph does not always antagonize heterochromatin formation/spreading. For example, demethylase Jumonji-containing-protein 2A (JMJD2A) is unable to demethylate H3K9me3 if H3S10 is phosphorylated , 75. In Drosophila melanogaster, H3S10ph was not upregulated in the genes activated in response to the heat shock, nor was it needed for the transcription of those genes in H3S10 kinase mutant [Of note, even though H3S10ph facilitates transcription, it is not a prerequisite for the transcription to occur. In stress-induced system in e mutant , 78. TheTaken together, H3S10 phosphorylation regulates transcription by: (1) promoting recruitment of chromatin proteins that support active transcription; (2) shielding gene regions from heterochromatin factors; (3) dictating the hierarchy and landscape of subsequent histone post-translational modifications; (4) contributing to the formation of binding platform for effector proteins.CCND1 gene promoter activity [BRCA2 are more sensitive to AURKA inhibitors [Deposition of H3S10ph mark during mitosis is mediated by members of the Aurora kinase family, comprising three highly homologous serine–threonine kinases which expression and activity peaks in M phase Table . Prelimiactivity . Also geactivity  but incractivity . On the activity . AURKA ihibitors .Table 1Hc-MYC, c-FOS, c-JUN, FOS1L, EGR1, that are responsible for transcriptional reprogramming of cells exposed to environmental stress-inducing stimuli. Since many of these IEGs are oncogenes, and H3S10 phosphorylation is typically triggered by factors known to be potentially carcinogenic, such as exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) [Interphase phospho-H3S10 mark is deposited on actively transcribed genes by multiple kinases (Table te (TPA) , 97, forte (TPA) , cigarette (TPA) , ionizinte (TPA) , H3S10phte (TPA) , 100. Cote (TPA) . An incrte (TPA) . H3S10phte (TPA) . The MSKte (TPA) . Subsequte (TPA) , 99, 100−/− mouse embryonic fibroblasts. These results show that RSK2 plays an important role in cellular transformation induced by tumor promoters such as EGF and TPA [RSK2, another MAPK-pathway kinase regulated downstream of ERK1/2, plays similar function to MSKs as it is able to phosphorylate H3S10, albeit with fourfold lower activity for this substrate . Stimula and TPA . Importa and TPA . Interes and TPA . In addi and TPA . JNK-med and TPA , formald and TPA  and arse and TPA .IEGs seem to be especially dependent on H3S10 regulation, as another H3S10 kinase, CDK8 was also identified as a positive regulator of IEGs. CDK8 is a part of mediator complex with a role in gene-specific regulation of transcription, thus functioning as a bridge between basal transcriptional machinery and transcription factors targeting specific genes. During serum-stimulation CDK8 is required for the proper transcription of IEGs, including oncogenic transcription factors . CDK8-decis bringing enhancer and promoter region via Mediator complex to promote assembly of transcription factors and RNA pol II on the target mRNA promoter sites. Mediator with CDK8 phosphorylates H3S10 to activate transcription. Knocking down a given ncRNAa resulted in a specific decrease in H3S10 phosphorylation at its target gene [Kinase activity of CDK8 towards H3S10 is regulated by specific class of lncRNAs, called ncRNA-activating (ncRNAa). Long ncRNAas act in get gene . This loget gene . Similarget gene –117. Besget gene . These r. In addition, CDK8 can modulate transcription in a very precise, signal-specific fashion. For example, after strong p21 activation, CDK8-mediator complex is selectively recruited to p53-target genes, augmenting p53 transcriptional program [Modulation of histone marks/chromatin is not the only mechanism by which CDK8 can regulate transcription. It can do so directly via phosphorylation of many transcription factors, including, among others, E2F1, NOTCH, p53, SMADs and STATs program . Similar program –121. In  program . Likewis program .FOSL1 and ID2. There, PIM1 catalyzes phosphorylation of H3S10 in the nucleosomes at MYC-binding site what enables transcriptional activation of those genes. MYC and PIM1 colocalize at the sites of active transcription, and together regulate 20% of the MYC-target genes. Consistent with this, PIM1 inhibition reduced cellular transformation potential of MYC [PIM1 is another interphase H3S10 kinase with a documented role in cancer initiation and progression. Interestingly, PIM1 overexpression alone is not sufficient to induce tumorigenesis with high penetrance. In marked contrast, simultaneous overexpression of PIM1 and MYC dramatically accelerates MYC-induced lymphomagenesis, leading to formation of aggressive lymphomas in utero or around birth –128. Aftl of MYC .In addition to the role in IEGs expression, H3S10 phosphorylation is required for the proper transcriptional response to proinflammatory signals activating NFκB, such as lipopolysaccharides (LPS), CD40 and tumor necrosis factor (TNF) –131. AltGiven the importance of H3S10ph in mediating/maintaining tumor cell phenotype, oncogene transcription, inflammatory signaling pathways, cancer-related inflammation and immune escape, modulation of the deposition of this mark has a broad therapeutic potential. In addition, since kinases capable of H3S10 phosphorylation are able to regulate a wide range of substrates beyond H3S10, their inhibition has broad, pleiotropic effects. This pleiotropy makes them very attractive therapeutic targets, promising multidirectional and multi-layer activity. In fact, many of the enzymes catalyzing phosphorylation of H3S10 have long been considered targets for cancer therapy , 137–141Phosphorylation of Serine 10 on histone H3 is a dynamic histone posttranslational modification and an important epigenetic event that affects chromatin activity during numerous biological processes. It is responsible for the hierarchy of histone modifications involved in the formation of different protein binding platforms, inducing transcriptional activation by promoting histone acetylation, blockade of repressive epigenetic information and shielding gene regions from heterochromatin spreading. During cell division, this mark is essential for chromosome condensation and segregation. It is also involved in the function of specific DNA-RNA hybrids, called R-loops Fig. .Fig. 2EfDeregulation of H3S10ph seems to have a major effect on the cellular transformation process. Many of the enzymes catalyzing H3S10 phosphorylation and dephosphorylation are long-known oncogenic factors. Traditionally, they were thought to affect gene expression through the modulation of signaling pathways and the activity of transcription factors. Now, with more reports on the role of H3S10ph in tumor cells, it becomes clear that their aberrant activity can lead to more profound changes in the chromatin structure. An important aspect of the epigenetic regulation is its potential long-lasting effect, that, through the mechanism of epigenetic memory, can be transmitted during mitosis. It ensures long-term maintenance of the results of the activity of those enzymes, even after initial developmental and environmental stimuli have been removed. Thus, surveying chromatin changes should be incorporated during development of therapeutic compounds aiming at inhibition of those enzymes.Given the broad substrate spectra of these kinases, the role of alterations in H3S10ph-associated chromatin structure/function triggered by their inhibitors is likely responsible for only a part of their anti-tumor activity; importantly, this part has not been broadly addressed and remains largely unknown. As the H3S10-regulated transcriptional activity is generally confined to specific gene sets, deconvolution of transcriptional program(s) disturbed by a given H3S10 kinase inhibitor would greatly improve understanding of their mechanism of action, and—more importantly—guide rational combinations with other molecularly directed drugs, immunotherapeutics, or conventional chemotherapy. Discovery of other links between chromatin remodeling and signaling pathways will help develop more precise, knowledge-driven therapeutic strategies.


Enterococcus mundtii QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure l-lactic acid (≥ 99.9%) via homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar , termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of E. mundtii QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of talC, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates. Optically pure lactic acid is a feedstock for the production of poly-lactic acid (PLA), a biobased and biodegradable plastic , and of Enterococcus mundtii QU25 is a non-dairy lactic acid bacterium, isolated from ovine feces that can ferment both cellobiose and xylose to produce optically pure l-lactic acid (≥ 99.9%) via homo-fermentation .,12.11,12cre/CcpA complex. These data are consistent with our previous results, which demonstrate a gradual reduction in the expression of several genes in the xylose operon with the increase of the glucose concentration in the glucose-xylose mixture [We then examined the expression of genes involved in xylose metabolism . The exp mixture .In spite of the simultaneous consumption of cellobiose and xylose, the transcriptional level of the initial xylose metabolism genes was approximately 7-fold lower in the cellobiose-xylose mixture than that in the xylose samples (ccpA and hprK (encoding HPr kinase/phosphorylase) were constitutively expressed under all conditions tested, the expression of ptsH (HPr) was approximately one-fourth times lower in the samples of the glucose-xylose mixture compared to that in the xylose samples , histidine-containing protein (HPr) (EMQU_0954), and HPr kinase/phosphorylase (EMQU_1951) ,12. WhilTransketolase and transaldolase are key enzymes in the PP pathway, and support homolactic fermentation from pentoses in QU25 . In the talC (EMQU_2814), which degrades sedoheptulose 7-phosphate (S7P), was highly induced by xylose; while very low amounts of the talC transcript were detected in the cells grown in glucose, cellobiose, glucose-xylose, and cellobiose-xylose media compared to those in xylose  sequence was identified at position -1078 upstream of talC (EMQU_2814) start codon, and was located in the coding region of an adjacent gene (EMQU_2815) predicted within the same operon, suggesting that talC expression in QU25 is likely repressed by CcpA-mediated CCR. In the cellobiose-xylose mixture, the amount of the talC transcript appeared to be insufficient for transaldolase enzymatic activity.The only annotated gene that encodes transaldolase, n xylose . A putatEscherichia coli to allow for the metabolism of xylose [via S1,7P by the universally conserved glycolytic enzymes, ATP-dependent phosphofructokinase  and aldolase . Genes encoding both of these enzymes  were highly expressed in the cellobiose-xylose mixture of QU25 cells , was mutated to achieve simultaneous consumption of glucose and pentoses to produce ethanol [Though lignocellulosic biomasses are one of the most abundant and sustainable resources, the development of methods to enable their utilization for biofuels and biobased materials has been slow. Since lignocellulosic hydrolysates contain both hexoses and pentoses, CCR is one of the difficulties hindering their use as substrates for microbial fermentations to produce both biobased materials and biofuels. Many attempts have been made to resolve this issue. For instance, in ethanologenic  ethanol . Further ethanol . However ethanol .Optically pure lactic acid is a biobased feedstock that is generally produced by homolactic fermentation with lactic acid bacteria from not only hexoses but also pentoses via the PP pathway. Among the facultative anaerobic Firmicutes, non-dairy enterococci have recently become a primary focus for application in the industrial production of lactic acid in America, Europe, Asia and Africa –25. SomeE. mundtii QU25 has been suggested as a candidate organism for the fermentation of lignocellulose due to its superior production of lactic acid and its ability to consume xylose in the presence of cellobiose during lactic fermentation. Though the most effective temperature for lactic acid production of QU25 was 43°C, this strain is able to grow at 50°C [The gene expression pattern in the whole genome of QU25 cells grown in the cellobiose-xylose mixture was more similar macroscopically to that of cells grown in xylose compared to that of cells grown in the glucose-xylose mixture . MoreoveFocusing on the xylose operon genes responsible for the incorporation and initial xylose metabolism including genes encoding xylose isomerase and xylulokinase, we demonstrated that the operon genes were still repressed in the cells grown in the cellobiose-xylose mixture compared to those in the xylose medium. However, the amounts of their transcripts were more compared to those in the glucose-xylose mixture , which atalC  complex acting as a general repressor for CCR had been supposed to be relieved in cells grown in cellobiose and xylose mixture , howeverssed see . TherefoClostridium acetobutylicum, it has been reported that the PRDs-containing transcriptional activator CelR and sigma factor σ54 (sigL product) regulated cellobiose utilization [celR genes (EMQU_0827 and EMQU_0874), ten PRD domain-containing genes (sigL (EMQU_2018) in the QU25 genome. In C. acetobutylicum, CelR binds to upstream activator sequences (UAS) at sites 80–150 bp upstream of the sigL promoter [sigL promoter located downstream of one of the celR orthologs (EMQU_0874), were highly induced by cellobiose  bind to the promoter region of the xylose operon and function in the upregulation of the xylose operon in the cells grown in the cellobiose-xylose mixture, in the presence of typical cre-CcpA-dependent CCR  and sometimes EIIA and EIIB domains, were previously reported as being involved in the CCR mechanisms . In Closlization . We idenng genes , and onepromoter , and in llobiose . Howeverdent CCR .E. mundtii QU25, for the fermentation of lignocellulosic hydrolysates by the simultaneous consumption of various sugars, which can be readily applied to industrial level production of optically pure lactic acid. Further studies to advance this work will include the development of a system for the genetic modification of this strain, similar to that previously reported for the successful engineering of microorganisms to overcome CCR. Next, we will attempt to delete the cre sequence upstream of the xylose operon to avoid binding the CcpA complex to the sequence, which serves as a repressor. We will also add a binding sequence for the putative positive transcriptional regulator activated by cellobiose, the existence of which in QU25 was predicted in this study.Herein, we have successfully described the use of S1 FigFermentation profiles of lactic acid production in cellobiose-xylose (A), glucose-xylose (B), glucose (C), cellobiose (D), and xylose (E) sugar mixtures.E. mundtii QU25 was cultured in a 1 L jar fermenter containing 360 mL mMRS medium at 43°C with 100 rpm agitation and at pH 7.0 (adjusted with 10 M NaOH). The sampling points for RNA-seq are indicated by arrows.(TIFF)Click here for additional data file.S2 Fig2 (100 mM), 2.5 μL of ATP (100 mM), and 2.5 μL of crude extracts at 37°C for 1 and 2 h. The filtered supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a LCMS-8050 triple quadrupole mass spectrometer system  with a reversed-phase Mastro SP column  using the same analytical conditions as previously described [Amounts of S7P and S1,7P extracted from the enzymatic reaction mixture were measured by LC-MS/MS. The crude extracts for the enzymatic assays were obtained from cells grown in xylose. The reaction mixture (50 μL) was incubated with 25 μL of MOPS-KOH buffer , 2.5 μL of ribulose 5-phosphate (25 mM), 2.5 μL of xylulose 5-phosphate (25 mM), 2.5 μL of MgClescribed . Quantif(TIF)Click here for additional data file.S3 Figcre, catabolite responsive elements; PRD-TA; PRDs-containing transcriptional activators. This illustration was created using BioRender.com.Abbreviations: IIA, IIB, IIC components of cellobiose-specific PTS; Cellobiose 6-P, cellobiose 6-phosphate; FBP, fructose 1,6-bisphosphate; CcpA, carbon catabolite protein A; HPr, histidine-containing protein; HPr-Ser-P, phosphorylated form of HPr; HPrK, HPr kinase/phosphatase; (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableThe data table consists of gene locus tag ID, predicted operon ID using Genome 2D, gene annotation from NCBI RefSeq and KEGG, RPKM values (Reads Per Kilobase per Million reads sequenced), and fold change values with corresponding p-values. Statistically significant fold change values are shown in bold . For the designations, such as X/G, see (XLSX)Click here for additional data file.S3 TableFor the designations, such as X/G, see (XLSX)Click here for additional data file.


Runx1+/− mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRβ rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.T-cell receptor gene beta (TCRβ) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRβ sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRβ rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRβ gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRβ gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the  RUNX1 is among the most frequently mutated genes in various hematological malignancies and RUNX1 alterations can lead to a loss of RUNX1 function or to a dominant-negative effect5. Mono-allelic RUNX1 mutations occur in approximately 15% of T-Cell Acute Lymphoblastic Leukemia (T-ALL), predominantly in cases with an immature phenotype and a poor prognosis8. In de novo Acute Myeloid Leukemia (AML) patients, somatic mutations in RUNX1 are detectable in approximately 3% of children and 15% of adults4. In an AML subgroup with an immature phenotype (AML-M0), 30% of the cases are associated with bi-allelic inactivating RUNX1 point mutations and deletions9. Patients with Myelodysplastic Syndrome (MDS) carrying RUNX1 mutations have a higher risk and shorter latency for progression to AML10.RUNX1 belongs to the evolutionary conserved Runt transcription factor family and is indispensable for the establishment of definitive hematopoiesis in vertebrates and is an important regulator of cells of the immune systemRUNX14. We focus in this study on the most common translocation, t, occurring in approximately 20% of childhood B-cell precursor (BCP) ALL patients12. This translocation results in an ETV6-RUNX1 fusion gene, encoding the N-terminal non-DNA binding moiety of ETV6 (12p13) fused to the almost entire RUNX1 protein coding region (21q22) including its DNA-binding Runt-domain (RHD), transactivation domain (TAD) and the VWRPY motif4. Twin studies have shown that the ETV6-RUNX1 translocation is the founder translocation in this BCP-ALL subgroup and is acquired in utero in very early progenitor cells prior to T- or B-cell receptor gene rearrangements13. Further genetic alterations leading to ALL can develop after years of latency13. Papaemmanuil and colleagues have characterized secondary events associated with leukemic transformation in ETV6-RUNX1 ALL, employing exome and low-coverage whole-genome sequencing. They found an enrichment of binding sites for the recombination activating gene (RAG) proteins in close vicinity to the genomic breakpoint junctions and concluded that RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 ALL14.Furthermore, there are over 50 different types of chromosomal translocations affecting 15. Thereby functional IG or TCR receptors are assembled from preexisting sets of Variable (V), Joining (J) and — in case of TCRβ, TCRδ and IGH — from additional Diversity (D) gene segments16. These segments are flanked by recombination signal sequences (RSS) composed of conserved heptamer and nonamer sequences separated by a spacer of 12 or 23 base pairs  and T-cell receptor (TCR) rearrangementsThe role of the ETV6-RUNX1 fusion protein was hitherto almost exclusively linked to the role of RUNX1 as a transcription factor investigated in more than 3000 publications. However, the RUNX1 DNA-binding core motif TGTGGNNN overlaps with the RSS heptamer and nonamer motifs which are crucial for the recombination of IG and TCR gene segments  and evaluated the number of B- and T-cells by immunohistochemistry (IHC). The characteristics of the mice used have been previously described .Functional TCR or IG rearrangements are essential for the survival of mature B- and T-cellsP < 0.0001) smaller in Runx1−/− mice  which was more pronounced in Runx1−/−  as compared to wildtype mice  and in Runx1−/− mice  compared to wildtype mice  in comparison to Runx1+/− and wildtype mice  in Runx1+/− mice and almost absent in Runx1−/− mice -based analysis . A mean of 503,199 joint sequence read pairs  with TCRβ V and J segment matches were generated resulting in a mean of 10,257  individual TCRβ gene rearrangements/clonotypes for each sample  in thymi and 4.4-fold (P < 0.0001) in the spleens of Runx1−/− in comparison to Runx1+/− and wildtype mice  lower ratio of functional to non-functional thymic TCRβ rearrangements in Runx1−/− mice compared to Runx1+/− and wildtype mice leading to a proportion of approximately 50% non-functional TCRβ rearrangements in Runx1 double knockout mice . In addition, absence of RUNX1 also affects transcription factor activity which might additionally contribute to a disturbed T-cell selection process.TCRβ richness was reduced 4.0-fold  thymic functional (ii) thymic non-functional (iii) splenic functional or (iv) splenic non-functional clonotypes  TCRβ CDR3 lengths were only observed in functional TCRβ rearrangements in the spleen, regardless of the Runx1 knockout status  longer N lengths in the thymi of Runx1 knockout mice  more extensive truncation at the 5′ end of the TCRβ D2 segment, leading to shorter TCRβ D2 segment lengths which was not observed for 5′ TCRβ D1  and J segments  in Runx1−/− mice compared to wildtype mice . . Fig. .Taken together, we found that Runx1 loss led to an overall reduction of the TCRβ repertoire and to an altered CDR3 architecture due to an increase of V, 5′ D2 and J segment nucleotide excisions and N nucleotide additions. An increased variability in CDR3 length was observed for both, functional and non-functional, TCRβ clonotypes indicating that RUNX1 directly affects VDJ rearrangement. A more pronounced truncation at the 5′ end of the TCRβ D2 segments correlated with lower TCRβ D2 segment usage and therefore a shift towards a higher thymic TCRβ D1/D2 segment ratio Fig. . The alt27. To further assess the role of Runx1 in the skewed TCRβ D1/D2 segment ratio in Runx1−/− mice, we analyzed by chromatin immunoprecipitation (ChIP) the capability of Runx1 to directly bind the TCRβ D1 and D2 segments which have been reported to represent the initiation sites of TCRβ rearrangements28. Runx1 ChIP enrichment was detectable for TCRβ D1 and D2 Runx1 heptamer sequences as well as the Rag1 core promoter (positive control) whereas no binding was found for the CD19 promoter (negative control)  Fig. . The binwww.ensembl.org), we found the RUNX1 heptamer motifs at the 5′ borders of TCRβ D1 and D2 segments to be highly conserved in Mammalia and Eutheria, respectively  genome, used for comparison as a more distantly related outgroup, harbored only a TCRβ D1 segment with a CACCGTG heptamer sequence with no overlapping RUNX1 sites. Compared to the highly conserved 5′ TCRβ RUNX1 heptamer motifs, the 3′ TCRβ D1 and D2 segments showed more variability and convergent evolution of RUNX1 heptamer motifs as exemplified by the CACAATG heptamer motif overlapping with a RUNX1 binding site at the 3′ TCRβ D1 segment of primates and the murine and rat TCRβ D2 segments  4,671,973 heptamer hits (+/−7 nucleotides) and (ii) 167,320 heptamer hits (+/−7 nucleotides) within RSS modules were used  in 65 heptamer hits and 3.7-fold overrepresentation (Z-score 5.0) within 15 heptamer hits within RSS modules. A Z-score > 2 is significant compared to normal background distribution, which can be assumed for sufficiently large data sets. This is the case for the dataset with all heptamer regions but not for the heptamer regions in RSS models. However, for the latter, the probability of finding the observed RUNX1 hits is P < 0.00004 based on a binomial distribution  closer to the deletion border (median 6 bp) as compared to RUNX1 motifs without an adjacent spacer-nonamer (median 36.5 bp). The genomic positions of heptamer, nonamer and RUNX1 motif hits at the borders of the 56 deletions are given in Supplementary Table Next we analyzed the spatial distribution of RUNX1 heptamer hits at the border of the 56 recurrent deletions in CDKN2A/B breakpoint we performed an EMSA29. In the presence of Molt-4 nuclear extracts containing wildtype RUNX1, a shift in the mobility of the RUNX1 motif oligonucleotide was detectable. This mobility shift was strongly reduced by adding an unlabeled competitor and was completely abolished by using a mutated RUNX1 binding motif altered at 3 nucleotide positions Using these 1,931 published ChIP-Seq peaks, we performed an enrichment analysis for overlapping RUNX1 motifs with heptamers associated with RSS modules and found 30 ChIP-Seq peaks associated with RSS modules. 15 of these 30 ChIP-Seq regions displayed RUNX1 motifs overlapping with the heptamer, compared to an expected number of 7.6 ± 2.7 SD, derived from the whole genome control. This represents a 2-fold enrichment  were compared to 11 RUNX1 wildtype T-ALLs, matched for an immature (IM) stage of maturation arrest, as defined by the absence of cytoplasmic or surface TCRβ protein expression31. The wildtype T-ALLs were selected to have at least incomplete clonal TCRβ rearrangements, to enable comparison of TCRβ D1 vs. D2 segment usage consisting of 5 T-ALLs with clonal TCRγ rearrangement (IMG) and TCRβ DJ rearrangement and 5 T-ALLs with a clonal TCRβ VDJ rearrangement (IMB) on at least one allele , 3 IMG, 2 IMB, 2 pre-AB T-ALL (which expressed cytoplasmic but not surface TCRβ protein) and one which expressed a surface TCRγδ receptor (TCR-GD+). With regard to TCRβ rearrangements, the 13 RUNX1 mutated T-ALLs demonstrated 12 clonal TCRβ rearrangements in 8 patients, including 9 TCRβ DJ and 3 TCRβ VDJ rearrangements. As expected, the latter were found in the T-ALLs with a more mature stage , including 11 TCRβ DJ and 7 VDJ rearrangements. Of these 18 rearrangements, 11 (61%) used the TCRβ D1 segment, which was not significantly different from the TCRβ D1/D2 segment usage in RUNX1 mutated T-ALLs (P = 0.25).To evaluate the impact of heterozygous RUNX1 mutated cases used the TCRβ J1 cluster in 6/12 (50%) cases and RUNX1 wildtype in 8/18 (44%) which was not significantly different (P = 1.0). There were too few clonal rearrangements to consider TCRβ CDR3 length or extent of 5′/3′ D segment truncations.Regarding TCRβ J segment usage, RUNX1 mutated T-ALLs which undergo TCRβ rearrangement is lower  compared to T-ALL in general where over 80% are rearranged, as previously reported31. No significant difference could be detected for the TCRβ D1/D2 or J1/J2 segment usage in RUNX1 mutated compared to RUNX1 wildtype human T-ALLs.Taken together, the proportion of 16. The excision of these DNA regions can be regarded as “physiological deletions”. This deletion process needs to be tightly controlled since misguided activity of the recombinase machinery might disturb the composition of the T-cell repertoire or – if directed against other genomic regions – many biological functions including cell proliferation and differentiation. At the core of the recombination machinery are the RAG proteins15. The RAG gene locus, comprising RAG1 and RAG2, has been reported to be coordinately regulated by RUNX proteins and we confirm here Runx1 binding to the Rag1 core promoter33. The transcription factor RUNX1 is also involved in the regulation of T-cell-specific genes such as CD4 and CD834. Only recently an additional function for RUNX1 as a recombinase cofactor has been reported. By interaction with RAG1, RUNX1 leads to enhanced deposition of RAG1 to the human TCRδ D2 segment18. However, it remained unknown if RUNX1 also acts as a recombinase cofactor for TCRβ gene rearrangements or if this activity can be usurped to initiate non-physiological deletions.Generation of functional TCRs requires the recombination of V, (D) and J TCR gene segments with excision of the intervening DNA sequences and the addition of random nucleotides at the V(D)J junctionRunx1 knockout mouse model combined with a deep sequence analysis of TCRβ gene rearrangements. In this mouse model an in-frame deletion of exon 5 of the Runx1 DNA-binding Runt domain was achieved in hematopoietic stem cells by Cre expression under the vav regulatory elements21. We demonstrated that in wildtype mice Runx1 is expressed in the sub-capsular region of the outer thymic cortex where TCRβ rearrangements are initiated and that the complete loss of Runx1 led to a highly disturbed thymic architecture and 4.8-fold reduction of mature CD3 positive T-cells. Interestingly, heterozygous loss of Runx1 had little or no impact for the thymic architecture or T-cell content. In contrast, Runx1−/− mice demonstrated an approximately 4-fold reduced TCRβ richness indicating that the absence of Runx1 leads to less effective TCRβ gene formation. This T-cell lymphopenia in Runx1 knockout mice is thus likely attributable to the absence of the recombinase cofactor activity of Runx1. Especially as it has previously been published, in a RunxF/FLck –cre mouse model, that upon Runx1 knockout a block in T-cell development occurs at the transition of the CD4/CD8 double negative (DN) stage 3 to DN4, that depends on assembly and expression of functional TCRβ rearrangements34. However, a contribution of the Runx1 transcription factor activity for the reduced T-cell number cannot be ruled out.To investigate the role of RUNX1 for TCRβ rearrangements, we employed a 28. Indeed, our ChIP analysis revealed that Runx1 binds to murine TCRβ D1 segments at the 3′ Runx1 heptamer motif and to TCRβ D2 segments matching with 5′ and 3′ Runx1 heptamer motifs. The RUNX1 heptamer motifs at the 5′ borders of TCRβ D1 and D2 segments are highly conserved in Mammalia and Eutheria, respectively. Notably the zebrafish genome used for comparison as a more distantly related outgroup harbored only a TCRβ D1 segment with heptamers without overlapping Runx1 sites. Consistent with our model, Runx1 knockout in zebrafish, having no Runx1 binding site overlapping its TCRβ D segment heptamers, was reported to have no impact on T-cell numbers and TCRβ rearrangements35.In the case that RUNX1 acts as a recombinase cofactor, its binding to the initiation site of TCRβ rearrangements at the D segment regions can be regarded as a prerequisite to induce excision at the TCRβ locusWithin Mammalia the occurrence of RUNX1 heptamer motifs at the 3′ borders of TCRβ D1 and D2 segments differs between species. A RUNX1 heptamer motif can be found at the 3′ border of the murine TCRβ D2 segment but not at the 3′ border of the human TCRβ D2 segment. Of note, we detected convergent evolution of RUNX1 heptamer motifs at the 3′ borders of TCRβ D segments (e. g. overlapping of the “CACAATG” motif with a RUNX1-binding site at the 3′ TCRβ D1 segment of primates and the murine and rat TCRβ 3′ D2 segment). Such convergent evolution strongly indicates a similar selection pressure in different mammalian species for the evolution of RUNX1 heptamer motifs adjacent to the TCRβ D segments.Runx1−/− mice compared to wildtype mice. Notably these alterations were also observed in non-functional TCRβ rearrangement which are not affected by the TCR-MHC selection process. Furthermore, the CDR3 proportion is altered in the thymus of Runx1−/− mice by 1.2-fold longer N regions, a 1.2-fold more extensive truncation at the 5′ end of the TCRβ D2 segment and a 2.5-fold reduction of TCRβ D2 segment usage. The more marked reduction in TCRβ D2 segment usage may reflect the presence of two Runx1 binding motifs compared to only one in TCRβ D1.The strongest evidence that RUNX1 directly impacts TCRβ CDR3 formation by its recombinase cofactor activity was provided by the observation of a higher CDR3 length variability in Runx1−/− mice.In essence, our data reflects the important role of RUNX1 for controlled and proper generation of the TCRβ repertoire and CDR3 composition. Thus, a malfunction of RUNX1 causing impaired TCRβ rearrangement is most likely also responsible for the high rate of non-productive TCRβ rearrangements (50%) observed in the thymi of Runx1 knockout mice. Significantly shorter (P < 0.0001) TCRβ CDR3 lengths were found only in functional TCRβ rearrangements of the spleen regardless of the Runx1 knockout status. This is in agreement with previous publications reporting longer CDR3 lengths in T-cells of the thymus as compared to post-thymic T-cells26. In murine models, Yassai and colleagues described this TCRβ CDR3 shortening as a molecular marker for positive selection in later stages of maturation of CD4 single positive thymocytes25. Therefore, the shortening of the functional TCRβ CDR3 length in spleen as compared to thymic T-cells indicates that a positive selection process takes place independent of the Runx1 knockout status. Thus, the accumulation of non-functional TCRβ VDJ rearrangements in the thymus of Runx1 knockout mice argues for an important role for RUNX1 in the TCRβ rearrangement process but not for the T-cell selection process.Interestingly, the composition of the rearranged TCRβ sequences was different in the thymi and spleens of Runx1−/− mice on the TCRβ architecture and T-cell repertoire, the TCRβ composition was essentially unaffected in Runx1+/− mice indicating that the presence of one intact allele is sufficient to exert the functions of RUNX1. This observation is in line with our finding that T-ALL patients with heterozygous RUNX1 mutations display no difference in TCRβ rearrangements as compared to matched immature T-ALL samples with no evidence of RUNX1 mutation.In contrast to the severe impact of the complete loss of Runx1 in Runx1−/− mice it was tempting to speculate that RUNX1 may under certain conditions also be involved in aberrant deletions events. To test this hypothesis, we investigated ALL cases with ETV6-RUNX1 rearrangements ) comprising approximately 20% of all childhood ALL12. In these patients the ETV6 protein dimerization domain is fused to the DNA binding and transactivation region of RUNX1. ETV6-RUNX1 ALL is characterized by a variety of additional most likely secondary chromosomal alterations which consist of recurrent and private deletions. These deletions predominantly occur in promoters and enhancers of genes involved in B-cell differentiation and it was shown that RSS and single heptamer motifs are enriched at the deletion sites14.Based on our finding that Runx1 impacts the physiological deletion processes in ETV6-RUNX1 ALL cases demonstrated the presence of RUNX1 heptamer motifs in close vicinity to these deletions14. A 2.4-fold overrepresentation  of RUNX1 binding sites overlapping with heptamers without adjacent nonamer motif and a 3.7-fold overrepresentation  of RUNX1 heptamer motifs within a RSS were found in ETV6-RUNX1 B-ALL deletion borders as compared to RUNX1 motif overlap with single heptamers and RSS backgrounds taken from the entire genome. Although this is not a functional proof, the significant enrichment of RUNX1 heptamer motifs at the deletion borders in conjunction with our Runx1−/− mouse TCRβ data clearly argues for an involvement of RUNX1 not only in physiological deletions but also for a major role for the generation of pathological deletions in ETV6-RUNX1 ALL.Our reanalysis of published data comprising 56 recurrent deletions derived from 37. Furthermore, our reanalysis of ChIP-Seq data derived from the ETV6-RUNX1 positive cell line REH suggests that ETV6-RUNX1 can bind to RUNX1 heptamer motifs in RSS of TCR and IG V-segments, indicating a role for the ETV6-RUNX1 protein in the ongoing TCR and IG rearrangements observed in ETV6-RUNX1 ALL30. In addition, our EMSA experiments with Molt-4 nuclear extracts provide evidence that the RUNX1 heptamer site at a recurrent, recombinase mediated, CDKN2A/B breakpoint described for ETV6-RUNX1 ALL is functionally relevant14. However, the data derived from our Runx1 knockout model also demonstrates that RUNX1 is not strictly essential for the physiological deletion process since TCRβ rearrangements still occur at very low frequency despite Runx1 knockout. This is consistent with our finding in ALL where - despite significant enrichment of RUNX1 heptamers at deletion borders - some deletion sites do not demonstrate RUNX1 heptamer motifs.Binding to the RUNX1 motif is not only possible for the native RUNX1 protein but also for the ETV6-RUNX1 fusion protein, which encodes almost the entire RUNX1 protein including its DNA-binding domain38. In this case the RUNX1 binding site overlapping with the heptamer is only separated by the RSS spacers of 12 or 23 bp length. Cieselak et al. already showed that RUNX1 and RAG1 can directly interact in the lymphoblastic cell line Molt-4 making it likely that RUNX1 can enhance RAG1/2 binding and initiation of deletions39.Therefore, we propose a model where RUNX1 (and/or ETV6-RUNX1) acts as an attractant for pathological deletion processes Fig. . However17. Moreover, RUNX1 may also act as a recombinase cofactor decisive for recurrent pathological deletions in ETV6-RUNX1 ALL by enhancing RAG binding at RUNX1 heptamer binding sites. Thus, RUNX1 needs to be taken into consideration as a recombinase cofactor for a better understanding of molecular mechanisms leading to physiological  and aberrant (e.g. ETV6-RUNX1 ALL) deletions.Taken together we propose a role of RUNX1 as recombinase cofactor in addition to its well-established function as transcription factor Fig. . In our Runx1fl/+-Tg(vav-Cre) X B6.Runx1fl/+ crosses21. . Only offspring carrying the Tg(vav-Cre) allele were used for analysis. The Cre recombinase is expressed under the Vav promoter, which is first expressed in the hematopoietic stem cells within the fetal liver, which give rise to all definitive hematopietic cells40. Cre expression inactivates the floxed Runx1fl allele by deletion of exon5, encoding the essential Runt DNA-binding domain. Consequently, all definitive (adult) hematopoietic cells in these mice will have a Runx1−/−. Runx1+/−, or wildtype Runx1 genotype.We performed TCRβ repertoire (NGS) and immunohistochemical analyses (IHC) on spleen and thymus tissues of littermates from B6.Runx1+/−, 10 Runx1−/−) with the age of 7 weeks, were analyzed. In addition, the thymi of 6–7 weeks old wildtype mice were used for Runx1 ChIP-analysis.All animal strains were maintained at the animal facility of the Heinrich-Pette-Institute. All animal studies were approved by the Hamburg commission for animal experiments (Nr. 89/13) and the LaGeS0 (T0114/05). All experiments were performed in accordance with relevant guidelines and regulations. In total 30 mice  the mean of the first two medians (e.g. wildtype and Runx1+/−) was employed to calculate the fold changes with the median of the other group (e.g. Runx1−/−).Immunostaining of thymus and spleen tissue was performed on consecutive sections, using the BondMax™ device (Leica Biosystems) employing the manufacturer´s protocol and reagents. For primary antibodies, dilutions of 1:500 were used for CD4  and CD19  antibodies. A dilution of 1:200 was used for CD8 , RAG1 , RAG2  and CD3  antibodies. The FOXP3 rabbit monoclonal antibody  was diluted 1:100. For antigen retrieval (20 minutes) the Bond-ER2 antigen retrieval solution (Leica Biosystems) was employed for all antibodies. The visualization of rabbit primary antibodies was performed by using the BOND Refine Detection System  including an anti-rabbit-HRP antibody and DAB/Hämalaun. The detection of rat primary antibodies was realized by using a rabbit anti-rat-bridge  followed by the BOND Refine Detection System as described above. The slides were evaluated independently by two pathologists. For quantification of CD19, CD3 and FOXP3 the mean of the two analyses was visualized as dot plots employing GraphPad PRISM V.6 software (GraphPad Software). Non-parametric analysis between data groups were performed by two-tailed Mann-Whitney U tests. The medians were used to calculate fold changes. If two groups were compared to another group (e.g. wildtype and 41. The primary PCR amplification (PA) were set up with AmpliTaq-Gold (Thermo Fischer Scientific), TCRsafe™ PA buffer (HS Diagnomics), 3.2 µM Primermix and 400 ng DNA template in a final volume of 50 µL. The cycling conditions were: 95 °C 12 min, 27 cycles of 95 °C 40 s, 64 °C 1 min, and 72 °C 1 min, with final extension at 72 °C for 7 min. In the second amplification (SA), adaptor sequences required for NGS were introduced, with Phusion High-Fidelity DNA Polymerase (Thermo Fischer Scientific), TCRsafe™ SA buffer (HS Diagnomics), with forward and reverse primers (1 µM each), in a final volume of 25 µL, using a 1:100 dilution of the corresponding primary PCR products as template. The cycling conditions were as follows: 98 °C 2 min, 5 cycles of 98 °C 20 s, 58 °C 30 s, and 72 °C 30 s, 5 cycles of 98 °C 20 s, 63 °C 30 s, and 72 °C 30 s, 13 cycles of 98 °C 20 s and 72 °C 30 s with final extension at 72 °C for 5 min. Spin column-purified PCR products were sequenced with MiSeq (Illumina) in paired-end mode (2 × 150 bp). For multiplexing standard TruSeq barcodes and MiSeq sequencing default conditions were employed allowing one mismatch in the barcode.Genomic DNA was extracted from formalin-fixed paraffin-embedded thymus and spleen tissue samples employing the Maxwell RSC FFPA DNA kit (Promega). A TCRsafe™ analysis was established to quantitatively analyze the murine TCRβ repertoire . In brief, read pairs delivered by the Illumina sequencer in paired-end mode were joined using the phred-like quality values per base to deliver a proper consensus sequence per single read pair. The joined reads were clustered and classified with respect to the TCRβ V and J segments as previously describedhttp://www.imgt.org/IMGTrepertoire) in combination with ensemble Blat searches for the C57Bl/6 mouse reference genome (https://www.ensembl.org).Noteworthy the TCRβ analysis was focused on all functional TCRβ alleles present in the C57Bl/6 mouse line used in this study. The TCRβ reference sequences for the C57Bl/6 mouse line were derived using the IMGT data base : The total number of clonotypes (richness) was calculated and for each TCRβ rearrangements the truncation of V, J and D segments and the length of N1 and N2 and the CDR3 region was determined. The ratio of functional/non-functional TCRβ rearrangements and the ratio of TCRβ D1/D2-segment usage was assessed.To analyze the impact of Runx1+/− compared to Runx1−/−) the mean of the two medians of the first group was employed to calculate the fold change between the two groups.Results were visualized as dot plots employing GraphPad PRISM V.6 software. Non-parametric analysis between data groups were performed by two-tailed Mann-Whitney U tests. The medians were used to calculate fold changes. If two groups were compared to on other group . Fresh-frozen mouse thymus was thawed on ice and cut into small pieces with a scalpel. The tissue parts were resuspended in 1.5 ml of ice-cold PBS (containing protease inhibitor) and incubated after addition of 150 μl of 11% formaldehyde solution at room temperature for 10 minutes. Quenching was achieved by adding 86.8 μl of 2.5 M glycine. The tissue was homogenized by a douncer followed by filtration through a 40 μm cell strainer. The filtrate was washed with PBS and cell amount was determined. Finally, approximately 2 × 10Rag1 core promoter. The B-cell specific CD19 promoter and a 3′ region of the Prame gene was used for comparison. Real-time DNA-PCR was performed with PowerUp SYBR Green Master Mix (Biosystems) on a Step One Plus Real-Time PCR Systems (Thermo Fisher Scientific) using the PCR parameters recommended by the manufacturer.ChIP enrichment of DNA fragments was determined by real-time DNA-PCR with primers covering the Runx1 binding sites in the TCRβ D1 and D2 heptamer sequences as well as the 44. All primers employed were tested to display an amplification efficiency of approximately 100% (+/−10%). Primer sequences are available from Supplementary Table Relative quantification of real-time DNA-PCR results was calculated using the comparative ΔΔCT methodwww.ensembl.org) using the murine TCRβ D1 and D2 segment with their adjacent RSS (including the spacers and nonamer motifs) as query sequences. As modification, the D2 segment with adjacent RSS from Notamacropus eugenii was used to detect the D2 segment in the genomes of Sarcophilus harrisii, Monodelphis domestica and Ornithorhynchus anatinus. The TCRβ D1 and D2 sequences were aligned manually. We employed Genomatix Software Suite v3.10  to determine RUNX1 binding sites (RUNX1 matrices V$AML1.01 and V$AML1.02 with a similarity threshold of 0.8) and Consite (http://consite.genereg.net/cgi-bin/consite) employing default conditions to predict C-FOS binding sites.To analyze if the RUNX1 binding sites adjacent to the murine TCRβ D1 and D2 segments are evolutionary conserved, we performed BLASTN searches 46.To address the question if a RUNX1 heptamer fingerprint marks the borders of recurrent 45. Likewise, two nonamer positional weight matrices were created  for heptamer sequences within RSS module hits and (ii) for all heptamer hits. For defining module matches in the deletion borders and in the genomic background, Genomatix ModelInspector was used47.The sequences extracted from the whole genome were used as background to calculate an overrepresentation of RUNX1 at the deletion borders of ETV6-RUNX1 positive ALL cell-line REH30. We focused on the determination of RSS within the ChIP-Seq peaks, employing Genomatix ModelInspector to identify module matches and subsequently performed an enrichment analysis for RUNX1 motifs overlapping with heptamers within RSS modules47.With the same methods described in the previous paragraph, we reanalyzed 1,931 published ChIP-Seq peaks of the CDKN2A/B locus29. These oligonucleotides harbored the RUNX1 heptamer binding site in the middle  were synthesized (Sigma-Aldrich) with the sequence of a published recurrent deletion border at the dle Fig. . The twodle Fig. . The corNuclear extracts were prepared from the Molt-4 T-ALL cell line employing NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher) and stored at −80 °C after snap freezing with 10% glycerol in liquid nitrogen.2, 75 ng/mL Poly(dI-dC), 0.05% NP-40, 2.5 µg/µl BSA, 4 µg nuclear extracts and 10 fmol biotin-labeled probe in a total volume of 20 µl. For competition the unlabeled probe was added to the reaction mixture prior to addition of the labeled probes with an excess of 400-fold molar concentration. After binding reaction, the samples were gently mixed with 5 µl 5X loading buffer and loaded onto a 6% DNA-retardation gel (Invitrogen). Gel running was performed using 0,5X TBE buffer and 110 V. Blotting was realized by using a 0.45 µm Biodyne B nylon membranes (Thermo Fisher), a XCell SureLock Mini-Cell system (Invitrogen) and blotting time of approx. 45–60 minutes at 100 V with 0.5X TBE buffer. Subsequently, UV-crosslinking of the membrane was done at 120 mJ/cm2 for 45 sec prior to chemiluminescence detection using HRP substrate solution (Thermo Fisher) and FusionCapt Advance analysis Software .Binding reactions were performed using the Lightshift Chemiluminescent EMSA Kit (Thermo Fisher) according to the manufacturer’s instructions with the following specifications: 1X binding buffer, 2.5% glycerol, 5 mM MgCl49. The multicentric trials were registered at ClinicalTrials.gov  and approved by local and multicenter research ethical committees. All experiments were performed in accordance with relevant guidelines and regulations. TCRβ rearrangements and their CDR3 junctions were compared in 13 T-ALLs with mono-allelic, acquired RUNX1 mutations and 11 T-ALLs with RUNX1 wildtype status. The RUNX1 mutation status for these cases was known due to a previous study50. TCR rearrangements were analyzed by Genescan analysis after EuroClonality multiplex PCR and by next generation amplicon sequencing of TCRβ DJ and VDJ rearrangements followed by Vidjil bioinformatics analysis53. Two-tailed Fisher exact tests were performed to compare the characteristics of the TCRβ rearrangements between the two groups.T-ALL samples were obtained at diagnosis from protocol treated children and adults for oncotyping and minimal residual disease quantificationSupplementary_Data.Supplementary_Table_S1.Supplementary_Table_S3.Supplementary_Table_S4.


Drosophila melanogaster that allows the simultaneous comparison of an enhancer’s ability to activate proximal and distal fluorescent reporter genes. Testing a panel of fluorescent transgenes in vivo, we found a two-protein combination that allows simultaneous measurement with minimal detection interference. We note differences among four tested enhancers in their ability to regulate a distally placed reporter transgene. These results suggest that enhancers differ in their requirements for promoter interaction and raise important practical considerations when studying enhancer function.Enhancers activate gene transcription in spatial and temporal patterns by interactions with gene promoters. These elements typically reside distal to their target promoter, with which they must interact selectively. Additional elements may contribute to enhancer-promoter specificity, including remote control element sequences within enhancers, tethering elements near promoters, and insulator/boundary elements that disrupt off-target interactions. However, few of these elements have been mapped, and as a result, the mechanisms by which these elements interact remain poorly understood. One impediment is their method of study, namely reporter transgenes in which enhancers are placed adjacent to a heterologous promoter, which may circumvent mechanisms controlling enhancer-promoter specificity and long-range interactions. Here, we report an optimized dual reporter transgene system in  A seconDrosophila (D.) melanogaster and test this system with four different enhancers, two heterologous promoters, and one endogenous promoter region.The broad relevance of tethering elements and enhancer-embedded remote control elements remains unknown, as these sequences are seldom sought out, and not identifiable by conventional methods. Typically, reporter transgene assays test an enhancer sequence placed immediately 5′ of a minimal heterologous promoter for which no choice of promoter is provided  cassette flanked by EcoRI and AscI restriction enzyme sites, and a 2,014 bp cassette flanked by AscI and AgeI sites. The EcoRI-AscI cassette possessed the DsRed.T4-NLS reporter transgene  and 3′ polyadenylation signal (flanked by EcoRI and FseI sites). The AscI-AgeI cassette contained the core region of the enhancer known as the dimorphic element  spacer sequence taken from the bab1 1st intron for which every other bp was replaced with a non-complementary transversion mutation, and an Hsp70 promoter with 5′ BglII and BamHI sites and 3′ XhoI and AgeI sites. The mS3aG vector was opened at its EcoRI and AgeI sites and the two synthesized cassettes were inserted to complete our first-generation red light/green light vector where the EGFP-NLS reporter gene was displaced from the dimorphic element enhancer by 1 kb.The vector backbone used for construction of the dual reporter system was the mS3aG reporter vector  of a second 2 kb spacer with flanking BglII sites into the BglII site of pRLGL2+DEcore. The second 2 kb spacer sequence was derived from a synthesized tandem duplicate 1 kb sequence from the bab1 1st exon, where the first and second kb of sequence were respectively altered at odd and even positions by non-complementary transversions, and for which all TTTAT (Abd-B binding motifs) were altered to TTGGG. The synthesized spacer piece was subsequently PCR-amplified using the primers In-Fusion (15) Spacer Fwd (CTGCCCGCCCAGATCTGGATTGTCAGCGTGTACACC) and In-Fusion (15) Spacer Rvs (ATGGCGCGCCAGATCTTCTCGGTACCCAATCTAATAAGC). The 8 kb spacer vector, pRLGL8+DEcore, was generated by In-Fusion cloning a synthesized 4 kb spacer sequence into the pRLGL4+DEcore vector at the BamHI site located at the 5′ end of the Hsp70 promoter for the DsRed.T4-NLS reporter gene. The spacer sequences were derived from a 4,000 bp sequence from the bab1 1st intron that had its nucleotide composition scrambled, substituted with non-complementary transversion at odd base pairs, had Hox-like binding motifs (YTAATKV and TTTAT) mutated, and that had restriction enzyme sites removed that are utilized as single cutters elsewhere in the pRLGL8+DEcore vector. The spacer sequence was synthesized (GenScript Inc.) and subsequently PCR-amplified for In-Fusion cloning using the primers InFus (15) 8k Fwd (CCGGCGCTCGGATCCGCTTTCTTAAGTAGTACGC) and InFus (15) 8k Rvs (CTCTGTAACTGGATCTTCAGTTCAAAACAATCGTTC).The 0 kb spacer version (pRLGL0+DEcore) was created by removing the 2 kb spacer from the pRLGL2+DEcore vector by BamHI and BglII digestion and subsequent re-ligation. A 1 kb spacer sequence was PCR amplified from the 2 kb spacer template using the RLGL2 1K spcF1 (TTTCCGggatccGCGCAACAACGCAGCTGGGTAGCG) and RLGL2 1K spcR1 (TTGCCagatctGTGTATCCGTCCCAGTACCTCG) primers which added flanking AgeI and SpeI sites of the S3aG  sequence of the tra gene (mCerulean-NLS) digestion and replaced by cassettes containing the coding sequences for other fluorescent proteins. This included the protein-coding sequence for mCherry, which was based upon the sequence in the pmR-mCherry vector (Clontech Inc.). The FASTA format sequence from the 1st codon of mCherry to the last codon amino acid was combined 5′ and in-frame of the coding sequence for the tra gene nuclear localization signal and a poly adenylation signal-containing 3′UTR. This coding sequence possessed single instances of StuI and SbfI sites, two restriction enzymes present in multi-cloning sequences of S3aG and pRLGL-type vectors. These sites were therefore removed by substituting a single synonymous base change. The E2-Crimson fluorescent protein (tra NLS sequence. The SbfI site that resided within the E2-Crimson sequence was destroyed by substituting a synonymous mutation. The mCerulean protein coding sequence was obtained from the CMV-Brainbow-1.0L vector (Addgene plasmid #18721), and the text was inserted in front of and in-frame with the text for tra NLS by AgeI and NotI restriction sites. These sequences were synthesized (GenScript Inc.) and cloned into the S3aG+yBE0.6 vector backbone after removal of the EGFP-NLS cassette, creating S3 am Cherry-NLS+yBE0.6, S3 am Cerulean-NLS+yBE0.6, S3aE2-Crimson-NLS+yBE0.6.The sequence between the the S3aG  vector cHsp70 promoter, E2-Crimson-NLS coding sequence, and a poly adenylation signal-containing 3′ UTR. This transgene was flanked by a 5′ BamHI site and 3′ EcoRI site. This sequence was synthesized and cloned in place of the distal reporter from the 2, 4, and 8 kb spacer vectors with the dimorphic element core enhancer. The 2 kb spacer was removed by BamHI and BglII digestion followed by re-ligation to create the version lacking a spacer. These E2-Crimson-NLS containing vectors are referred to as pFRGL0+DEcore, pFRGL2+DEcore, pFRGL4+DEcore, and pFRGL8+DEcore.A reporter gene sequence was designed for the pFRGL (Far Red/Green Light) vector composed of an D. melanogaster genomic DNA (strain 14021‐0231.04) that was acquired from the San Diego Drosophila species stock center. Enhancers were PCR-amplified from the genomic DNA using the primer combination shown in AscI and SbfI sites. These PCR amplified enhancers were cloned into the AscI and SbfI sites of the pRLGL8+DEcore vector in place of the dimorphic element core enhancer.The enhancer DNAs were obtained from Drosophila synthetic core promoter or DSCP  site and a 3′ AgeI (TTTGCCaccggtGGATCGTTTAAACAGGGCTCTCGAC) site. Following PCR and enzyme digestion, this promoter was ligated into the AscI and AgeI sites of the digested vector, creating the vector referred to as pRLGL8 w/o enhancer.To create a version of the pRLGL8 vector without (w/o) an enhancer, we digested the pRLGL8+DEcore vector with the AscI and AgeI restriction enzymes, which removed a cassette containing the dimorphic element enhancer and the Hsp70 promoter for the EGFP-NLS reporter gene, we used the AscI and AgeI digested pRLGL8+DEcore vector that lacked the dimorphic element enhancer and the Hsp70 promoter for the EGFP-NLS reporter gene. The dimorphic element was then PCR amplified with primers that added a 5′ AscI (TTCCGggcgcgccTCGCCTccgcggCTCTTTCTCTTTGCCATTTTAAC) site and a 3′ AgeI (TTTGCCaccggtGTGTGTGAACCAATTTGTTGTGC) site. Following PCR and enzyme digestion, this enhancer was ligated into the AscI and AgeI sites of the digested vector, creating the vector referred to as pRLGL8+DEcore w/o proximal promoter.To create a version of the pRLGL8 vector w/o the proximal D. melanogaster germline attP40 landing site (nd chromosome (Best Gene Inc.) by a phiC31 integrase approach  . Transgenic fruit fly lines are available upon request. Supplemental material available at figshare: https://doi.org/10.25387/g3.10299482.All of the relevant DNA sequences and the encoded fluorescent proteins can be respectively found in Supplementary Documents 1 and 2. All custom DNA sequences were synthesized by GenScript Inc. Plasmid vectors will be made available from the Addgene non-profit vector repository  gene in-frame with the coding sequence for the nuclear localization signal (NLS) of the tra gene on the 3′ end .In these Red Light/Green Light vectors, an enhancer can be cloned into the er genes . Both thHsp70 promoter is flanked by unique StuI and BamHI restriction sites, so this promoter can either be replaced by another promoter or supplemented with additional promoter proximal sequences. It should also be noted that the distal 3′ UTR can be removed by the flanking EcoRI and FseI restriction endonuclease sites in the event that another 3′ UTR or mutated version needs to be tested.The vectors with the added spacer sequence allow for the simultaneous testing of an enhancer’s regulatory activity on proximally and distally located promoters. Moreover, the distal Hsp70 promoter with the endogenous promoter of the gene that the enhancer regulates, adding endogenous promoter proximal sequence next to the distal promoter, or testing an expanded enhancer region.We initially sought to determine whether a particular enhancer can mediate its characteristic regulatory activity upon both proximal and distal reporter genes, resulting in equal reporter expression . It is aOnce a reporter configuration has been identified, where the regulatory activity is imparted effectively upon the distal reporter, it is then feasible to introduce enhancer mutations to map the sequences required for remote control element activity. For instance, a series of scanning mutant enhancers could be made where each mutant includes a unique block of base pairs that were altered by non-complementary transversions . These mHsp70 promoter of D. melanogaster (bric-à-brac (bab) gene complex . Importantly though, these data show that an 8 kb spacer sequence was suitable to interrupt the communication of the dimorphic element with a heterologous promoter in a D. melanogaster transgene system. However, DsRed.T4-NLS seems less than ideal as a reporter to use in conjunction with EGFP-NLS.In a typical reporter transgene, enhancers are placed immediately adjacent to a heterologous promoter, such as the nogaster . However complex  at vario complex -3F. Thisrescence ’’. Moreopression ’’-3F’’. pression ’’. This rescence ’-3F’. ThD. melanogaster.We were concerned that the inserted spacer sequences might possess unwanted enhancer activities that complicate the observed reporter expressions. Thus, we sought to see whether the 8 kb spacer  could drive reporter expression in the pupal abdomen when the pRLGL8 dual transgenes did not include an enhancer. This enhancer-less configuration did not drive any noteworthy expression in the pupal abdomen . While this observation does not rule out the possibility of the spacer possessing enhancer activities in other cell types or developmental stages, it is encouraging that this spacer may generally be lacking regulatory capability and be of broad use to the community studying gene expression regulation in Hsp70 promoter. The absence of this promoter resulted in an inability of the dimorphic element to activate EGFP-NLS expression, while no noteworthy expression activation was observed for DsRed.T4-NLS from the remaining distal promoter . These results suggest that promoter competition is not having unwanted effects on the utility of this vector system. Collectively, our results support the utility of this system for studying proximal and distal gene expression regulation simultaneously.A second concern was that placement of the DsRed.T4-NLS reporter gene’s promoter at a distance of 8 kb from the enhancer, created a situation where the enhancer could only activate one promoter at a time, and that there was an enhancer preference for the proximal promoter of the EGFP-NLS reporter gene. To test whether this concern is a real problem, we deleted the proximal Hsp70 promoter at an 8 kb distance, it remained a possibility that other enhancers possessed differing long-range activating abilities. Thus, we tested three additional D. melanogaster enhancers that are active during pupal development  enhancer of the gene promoter . In the promoter ’’’. Thispromoter ’’’. Thispromoter . It is pbab1 and bab2 genes , which is an element that resides in the intergenic region between the paralogous ectively . The LAEectively ’’’. Inteectively ’’. This Hsp70 promoter lacked an element or elements necessary for interacting with it. Thus, we replaced the distal Hsp70 promoter with the Drosophila synthetic core promoter (DSCP)  and DsRed.T4-NLS  fluorescent excitation and emission spectra distinct from EGFP-NLS, we sought to see whether it performs equally well in a dual reporter transgene context. Thus, we replaced the DsRed.T4-NLS coding sequence in the pRLGL0, 2, 4, and 8 kb vectors that possess the dimorphic element enhancer with the E2-Crimson-NLS coding sequence -7D. When element ’’-7E’’.  element ’-7E’, suDrosophila that permits the simultaneous comparison of an enhancer’s capability to activate a distal or proximal promoter sequence region. Using a well-studied enhancer involved in abdominal pigmentation, we found that this sequence can similarly activate two fluorescent reporter transgenes when they are at equal proximal positions. However, as one of the reporters is placed progressively further away from the dimorphic element (starting at 1 kb), the level of expression declines until it can no longer be observed (at a distance of 8 kb). Tests of three additional D. melanogaster enhancers revealed a range of capabilities to activate a distal promoter over long distances. Thus, different enhancers possess distinct capabilities to activate gene expression from a distally located heterologous promoter. For one tested enhancer its inability to activate the distal reporter gene at an 8 kb distance indicates that the enhancer’s in vivo function must be mechanistically complex, requiring sequences beyond its proximal promoter or enhancer-adjacent sequences. Using a combination of fluorescent proteins that we optimized for maximal spectral separation, this system will promote an understanding of the phenomenon of long-distance communication between enhancers and promoters.We have developed an optimized dual reporter transgene system in D. melanogaster bab locus as our test case. The endogenous function of this enhancer is to control the female-specific expression of the bab1 and bab2 genes in the A5-A7 segments of the pupal abdomen  for the ability to activate expression of a heterologous promoter in a reporter transgene assay. Our results suggest that this methodology is at risk for failing to identify regulatory activities when these sequences are at a distance to an ill-suited promoter. This justifies the additional examination of weak activities detected in larger genomic fragments, as the tested region may lack elements for long-range interactions. The existence of these features raises the conundrum that heavily dispersed elements that mediate long-range interactions may exist and be exceedingly difficult to find. Integrating these into a system such as Red Light/Green Light may require unbiased high throughput/genomic approaches such as Hi-C, 3C, and 5C . The curcis-acting sequences necessary for long-distance regulation. However, the LAE provides an opportunity to seek and characterize a remote control element. Future studies should subject the LAE to mutations to identify the RCE element as the sequence that when mutated results in attenuated distal reporter expression (Our motivation for developing Red Light/Green Light was to provide a means to identify the DNA sequences involved in mediating gene regulation between a distantly located enhancer and its target promoter. What has been previously referred to as remote control (pression . DiscoveA major theme in the evolution of development is that changes in gene expression, driven by non-coding mutations play significant roles in generating morphological traits . We sugg


Eukaryotic genes are packaged into dynamic but stable chromatin structures to deal with transcriptional reprogramming and inheritance during development. Chromatin remodeling factors and histone chaperones are epigenetic factors that target nucleosomes and/or histones to establish and maintain proper chromatin structures during critical physiological processes such as DNA replication and transcriptional modulation. Root apical meristems are vital for plant root development. Regarding the well-characterized transcription factors involved in stem cell proliferation and differentiation, there is increasing evidence of the functional implications of epigenetic regulation in root apical meristem development. In this review, we focus on the activities of chromatin remodeling factors and histone chaperones in the root apical meristems of the model plant species Arabidopsis and rice. Epigenetics refers to the study of heritable changes in gene expression that do not involve alterations to the nucleotide sequence. During plant growth and development, the chromatin structure in the nuclei must be precisely modulated in coordination with the transcriptional reprogramming associated with cell proliferation and cell fate determination within meristem regions. Nucleosomes, which are the basic structural and functional units of chromatin, comprise approximately 146 bp of DNA wrapped around a globular histone octamer, each containing two molecules of H2A, H2B, H3, and H4 . The strChromatin remodeling factors and their associated complexes can use the energy generated by ATP hydrolysis to regulate histone–DNA interactions and alter nucleosome composition, position, and occupancy, thereby altering DNA accessibility and functionally remodeling local or global chromatin structures ,7,8. In CHR719, corresponds to the Arabidopsis CHR12 and CHR23 genes [The genome of the model plant Arabidopsis encodes four SWI/SNF subfamily remodelers: BRM (BRAHMA), SYD (SPLAYED), and two structurally- and functionally-related proteins, MINU1/CHR12 and MINU2/CHR23 ,12. In r23 genes , implyinThe SWI/SNF ATPases have common domains in their N-terminus, including the QLQ (Gln-Leu-Gln) domain and the HSA (helicase-SANT-associated) domain, both of which are involved in the interaction between SWI/SNF ATPases and other proteins . A previPlant genomes also encode protein homologs of multiple required subunits of metazoan SWI/SNF complexes, including SWI3 homologs (SWI3A–D), SWP73-type proteins (SWP73A and SWP73B), ARPs (ARP4 and 7), and the SNF5-type protein BSH (BUSHY), as well as several optional proteins such as the OSA-type and POLYBROMO-type proteins . Gel filMany SWI/SNF chromatin remodeling factors can disassemble or slide nucleosomes in vitro . In yeasCHR11 and CHR17 genes, as well as a deficiency in the functional DDT domain proteins RLT1/2 (RINGLET1/2), result in impaired vegetative organ development and early flowering [In eukaryotes, ISWI proteins form conserved protein complexes with DDT (DNA-binding homeobox and different transcription factor) domain proteins. The resulting protein complexes can alter nucleosome positions without affecting the nucleosome structure  .The remolowering .The chromodomain is a unique protein domain containing conserved hydrophobic amino acids that interact with methylated lysine residues. The CHD subfamily chromatin remodeling factors have an additional double chromodomain before the Snf2 domain . Plants Compared with other chromatin remodeling factors, the INO80 subfamily proteins have a considerably longer “insertion region” separating the N-terminal SNF2_N and C-terminal Helicase_C. This subfamily includes the chromatin remodelers INO80 and SWR1 (SWI2/SNF2-related 1), both of which are highly conserved in eukaryotes and function as integrated scaffolds that interact with many other subunits to form protein complexes . Moreover, INO80-C shares many important and indispensable subunits with SWR1-C, such as RVB1 and RVB2, but there are also many complex-specific subunits, including ARP5 in INO80-C and ARP6 in SWR1-C . In eukaUnlike SWR1-C, INO80-C in eukaryotic organisms other than plants reportedly have diverse biological activities related to nucleosome eviction, establishment of nucleosome spacing, and removal of H2A.Z ,40. The Because of the strong electrostatic interaction between DNA and histones at physiological ionic strength, the orderly formation of nucleosome structures requires histone chaperones. These chaperones block non-specific interactions between positively charged histones and negatively charged DNA and are important for nucleosome assembly/disassembly, which are very dynamic processes in fundamental biological activities such as transcription and DNA replication, repair, and recombination .2 tetramer on the naked DNA by the corresponding chaperones, followed by the H2A-H2B histone chaperone-mediated addition of the H2A-H2B dimer on each side. This ordered process is reversed during nucleosome disassembly. Notably, H3-H4, but not H2A-H2B histones, can be deposited directly on naked DNA, which represents a critical control point for the formation of ordered structures in nucleosomes. Recent studies on nucleosome asymmetry and sub-nucleosomal structures, such as hemisomes/half-nucleosomes (one copy of H2A/H2B/H3/H4) and hexasomes (two copies of H3-H4 and one copy of H2A-H2B), indicate that multiple pathways may be responsible for the subtle regulation of nucleosome structures. Moreover, these sub-nucleosomal structures may be important for transcription [In eukaryotes, most histone chaperones are conserved regarding sequence and activity, and can be classified as H3-H4 or H2A-H2B chaperones. These two chaperone groups play specific roles in different nucleosome assembly/disassembly steps ,48. Histcription ,50.In plants, the most thoroughly studied histone chaperones with functions in the root apical meristem (RAM) are the CAF-1 (chromatin assembly factor-1) H3-H4 histone chaperone and the NRP (nucleosome assembly protein 1-related protein) H2A-H2B histone chaperones. Both were observed to assemble nucleosomes in vitro in earlier biochemical studies ,48,51.fas1 and fas2 mutants are recessive mutants that were isolated by forward genetics during the screening for mutations that lead to abnormal meristematic structures. Almost all of the organs of fas1 and fas2 mutant plants are affected, and the most obvious phenotype is the fasciation of plant shoots. The fas1 and fas2 mutants also exhibit decreased root growth [The H3-H4 histone chaperone CAF-1 was originally purified from the nuclear extract of human cells and was identified as a protein complex consisting of three subunits, p150, p60, and p48. The p150 subunit of human CAF-1 interacts directly with H3-H4 through its acidic region. This subunit also binds to PCNA (proliferating cell nuclear antigen), which recruits CAF-1 to replication- and DNA repair-coupled chromatin assembly sites ,53. The t growth . Both FAt growth ,56.Xenopus laevis eggs as a factor promoting nucleosome assembly in vitro [The NAP1 (nucleosome assembly protein 1) protein was first identified and purified from in vitro . The NAPin vitro . The NAPin vitro .The cell fate determination and pattern formation in meristems during organ development have always been key issues in studies of multicellular development. The root tip of the model plant Arabidopsis is an excellent research model because of its simple meristem organization, as well as the fact there are thoroughly analyzed Arabidopsis cell lineages with considerable genetic information. The SCN (stem cell niche) in the RAM is produced by the initial cells surrounding a number of relatively stationary stem cells that form the QC (quiescent center) . The surPLT expression levels promote stem cell maintenance, whereas intermediate expression levels enhance the mitotic activity in the RAM. Outside of the elongation zone, low PLT expression levels are required for cell elongation and differentiation [wox5 and plt mutants, implying these genes are important for determining the QC cell fate and the maintenance of the SCN.High-throughput experiments combined with bioinformatics analyses have established the transcriptome patterns in the RAM and generated some information regarding the transcriptional regulation in the RAM, thereby considerably increasing our understanding of the framework of the complete gene regulatory network during RAM development . A previntiation . The incbrm mutants have a defective root SCN and exhibit decreased meristematic activity and root growth retardation. Moreover, BRM maintains the root SCN by directly targeting the chromatin of several PIN genes (PIN1–PIN4 and PIN7) and altering their expression. Furthermore, the overexpression of PLT2 can partially rescue the root SCN defects in brm mutants [ABA INSENSITIVE 5 (ABI5), and the anchored BRM helps to stabilize a nucleosome that likely represses ABI5 transcription under non-inductive conditions. The brm mutants exhibit ABA sensitivity and increased drought tolerance, similar to the phenotype of ABI5-overexpressing transgenic plants [The SWI/SNF remodeling factor  mutants . Notablyc plants .IPT3/IPT7 (ISOPENTENYLTRANSFERASE 3 and 7), the key genes in cytokinin biosynthesis, and the CDK (cyclin-dependent kinase) inhibitor gene KRP7 (KIP-related protein 7) by inhibiting the deposition of the active histone mark H3K4me3 and chromatin loop formation, thereby controlling cytokinin production and cell cycle progression to maintain the RAM and promote root growth [The SWI/SNF complex SWP73-type subunit BAF60 can repress the transcription of t growth . NeverthMINU1/CHR12 and MINU2/CHR23 play redundant roles related to RAM establishment and maintenance. The phenotypes of the chr12 and chr23 single mutants are similar to the wild-type phenotype. In contrast, the chr12 chr23 double mutant, in which both genes are knocked out, exhibits plant embryo lethality and fails to produce embryonic meristems. Although a weak double mutant can germinate, the observed abnormal growth direction and the impaired rate of cell division result in a significantly defective RAM structure with an unrecognizable QC and an irregular columella cell layer [WOX5 promoter and repress expression. In contrast, the overexpression of CHR23 inhibits root growth because of decreased cell elongation [During plant development, ll layer . Additioongation .Chromatin remodeling and histone modifications are involved in many aspects of root development. In animals, CHD3 family members are included in the NuRD  complex, which helps repress transcription by coupling chromatin remodeling and deacetylation activities . Most stpkl mutants producing short roots with low root meristem activity [PKL result in the low penetrance of the “pickle root” phenotype, characterized by embryonic-like primary roots and the abnormal accumulation of seed storage reserves in the roots [SWN and EMF2 in roots. The deletion of PKL results in decreased PcG-mediated H3K27me3 levels and the increased expression of many PcG target genes in the roots, similar to the effects of mutations to PcG [An earlier investigation indicated PKL is necessary for establishing and maintaining the root meristem, with activity . Mutatiohe roots ,76. Theshe roots . Chromats to PcG ,79. Howes to PcG , indicatCHR729 regulates root growth through the GA (gibberellin) pathway. The bioactive GA3 content is reportedly decreased in the chr729 mutant, but the phenotype of this mutant may be partially recovered by the application of exogenous GA3 [CHR729 results in an overall decline in H3K27me3 and H3K4me3 levels, but not H3K4me2 and H3K4me1 levels [In rice, the PKL orthologous gene nous GA3 . Moreove1 levels .CHR11 and CHR17 or to the DDT domain genes RLT1/2 lead to the production of short roots [In Arabidopsis, double mutations to rt roots ; howeverrt roots , which rrt roots . A previrt roots . Althougrt roots , a similAs in mammals, H3 histones in plants are classified as the canonical histone H3.1 and the histone variant H3.3. Although these histones share similar sequences ,86, theiFAS1 and FAS2, which encode components of the Arabidopsis CAF-1 complex, result in inhibited root growth because of a decrease in mitotic activity as well as an abnormal SCN structure [fas1 and fas2 mutants exhibit chromosomal abnormalities during mitosis, such as the production of poly-centromeric chromosomes at metaphase as well as chromatid bridges and acentric fragments during anaphase-telophase [fas mutants [Mutations to tructure ,56,88. Telophase . A small mutants . In thes mutants , furtherfas1 and fas2 mutants, the identities of the initial cells are reportedly relatively undefined, and many initial cells exhibit diverse characteristics [Stem cells generate two daughter cells with different identities, allowing them to simultaneously self-renew and produce differentiated daughter cells. The influence of chromatin dynamics in this process deserves a detailed investigation. In the RAM of eristics ,92. Simieristics , demonstfas mutants also imply an early shift from mitotic activity to endoreduplication [fas mutants, resulting in a higher ploidy level of chromosomes, which is exemplified by enlarged epidermal cells and increased trichome branching on leaves [Double-strand breakage can promote the early initiation of endoreduplication by inducing the cell cycle G2/M arrest . The smalication ,56. Abnon leaves .NRP1 or NRP2 do not result in obvious changes in development, but mutants in which both genes are mutated exhibit a short-root phenotype during postembryonic development, with a cell cycle arrest at G2/M in the root tips as well as disordered cell organization in the SCN. The double mutant is highly sensitive to genotoxic stress, with elevated levels of DNA damage and a lack of transcriptional gene silencing, implying that NRP1/2 contribute to chromatin stability and meristem maintenance in developing roots [GLABRA2 (GL2), which is the WER downstream target gene that is critical for epidermal cell specification. The WER-dependent enrichment of NRP1 at the target promoter is accompanied by histone eviction and nucleosome loss, which is an example of a molecular mechanism mediating the recruitment of a histone chaperone by a gene-specific transcription factor to modulate the chromatin structure for proper cell differentiation [The NRP1/2 histone chaperones are specific to H2A-H2B, with both ubiquitously expressed in plant organs. Single mutations to ng roots . Recentlntiation .nrp1/2 fas2-4 double mutant, in which the middle subunit of CAF-1 is mutated, more than 8% of the Arabidopsis genes are incorrectly transcribed, including several genes related to root meristems, such as WOX5 and SHR (SHORT ROOT), and auxin-related ARF5 (AUXIN RESPONSE FACTOR 5) and IAA30 (INDOLE-3-ACETIC ACID INDUCIBLE 30). In the roots of the nrp1/2 fas2-4 triple mutant, the expression level of the auxin-response reporter gene fused to the green fluorescent protein (GFP) gene (DR5rev:GFP) was significantly downregulated, and the pattern of peak distribution at the QC was also largely lost. Thus, an abnormal auxin regulation pathway is likely responsible for the defective root development of the nrp1/2 fas2-4 mutant. Moreover, NRP1/2 and CAF-1 are considered to coordinately function to control chromatin replication and genomic integrity and prevent cell death in root meristems, which is essential for normal SCN functions [The H2A-H2B histone chaperone NRP1/2 and the H3–H4 histone chaperone CAF-1 play a synergistic role in SCN maintenance. The two chromatin factors coordinately regulate transcription. In the unctions .NRP1/2 and the chromatin remodeling factor INO80. The nrp1/2 ino80 double mutant exhibits an extreme short-root phenotype and hypersensitivity to the auxin transport inhibitor NPA. These two chromatin factors are recruited to the chromatin regions of the auxin efflux carrier gene PIN1, and mediate the local histone occupancy, thereby modulating the transcription level. Their combined functions are also implicated in chromatin replication and the maintenance of genomic integrity to ensure the viability of root meristem cells [A similar mechanism can also be found in the genetic interaction between em cells . HoweverCurrently, the studies on chromatin remodeling factors and histone chaperones in the RAM are mainly focused on replication-coupled chromatin stability and the transcriptional regulation associated with cell fate determination. However, because of the substantial abundance of chromatin remodelers and histone chaperones and their potential functional redundancies, many well-known factors are not associated with an obvious phenotype in the RAM. Future genetic analyses and investigations involving single-cell sequencing technology will help clarify the epigenetic pathways related to the initial cell specification and proliferation in the SCN and their differentiation. Moreover, other important issues still need to be resolved. For example, how chromatin factors specifically target genes and how they form complex regulatory networks will need to be determined. Future investigations of the chromatin remodeler–histone chaperone interactome and comprehensive chromatin immunoprecipitation sequencing analyses will be useful for elucidating the mechanism by which plant root tips precisely respond to environmental stimuli and developmental needs. Furthermore, the available techniques for in situ chromatin studies are critical for resolving the nuclear architecture . High re