Emulating nativetransient transcription machineries modulatingtemporal gene expression by synthetic circuits is a major challengein the area of systems chemistry. Three different methods to operatetransient transcription machineries and to modulate the gated transcriptionprocesses of target RNAs are introduced. One method involves the designof a reaction module consisting of transcription templates being triggeredby promoter fuel strands transcribing target RNAs and in parallelgenerating functional DNAzymes in the transcription templates, modulatingthe dissipative depletion of the active templates and the transientoperation of transcription circuits. The second approach involvesthe application of a reaction module consisting of two transcriptiontemplates being activated by a common fuel promoter strand. Whileone transcription template triggers the transcription of the targetRNA, the second transcription template transcribes the anti-fuel strand,displacing the promoter strand associated with the transcription templates,leading to the depletion of the transcription templates and to thedynamic transient modulation of the transcription process. The thirdstrategy involves the assembly of a reaction module consisting ofa reaction template triggered by a fuel promoter strand transcribingthe target RNA. The concomitant nickase-stimulated depletion of thepromoter strand guides the transient modulation of the transcriptionprocess. Via integration of two parallel fuel-triggered transcriptiontemplates in the three transcription reaction modules and applicationof template-specific blocker units, the parallel and gated transientlymodulated transcription of two different RNA aptamers is demonstrated.The nickase-stimulated transiently modulated transcription reactionmodule is applied as a functional circuit guiding the dynamic expressionof gated, transiently operating, catalytic DNAzymes. These CDNs systems were applied to assemble hydrogelmaterials revealing dynamic stiffness properties for self-healingand controlled drug release,39 operationof dynamic biocatalytic cascades40 andcontrolled catalysis, drug release, and functions of nanoparticles.42 In addition, dissipative, transient nucleic acid reaction frameworkswere applied for the dynamic, transient release of loads,43 operation of transient biocatalytic cascades,44 formation of nanostructures, and control overoptical properties of nanoparticles.46Transient interactions betweentranscription factors or hormones and the transcription machineryplay key roles in the dynamic modulation of gene expression49 transcriptional switches,50 bistable regulatory networks,51 programmedswitchable transcription within topologically constrained transcriptiontemplates,52 and constitutional dynamicnetworks guiding dynamic gene expression and protein translation.53 Moreover, with the advances to construct artificialcells, i.e., “protocells”,56 the need tointegrate transcription machineries into cell-like containments andto dictate complex spatiotemporal dynamic transcription processeswithin the artificial cell is a challenging goal. Indeed, recent studiesreported on the integration and activation of a transcription machineryin a fused liposome assembly57 and thetriggered topologically switchable operation of a transcription templatewithin a hydrogel microcapsule, cell-like containment.58 All of these processes are, however, far beyondthe desired spatiotemporal transcription complexity where transientgated, fan-out, and cascaded transcription processes are desirable.Emulatingthe dynamic features of native transcription machineriesby synthetic nucleic acids is still in its infancy, yet progress toadvance facets related to dynamic transcription and gene expressionhas been demonstrated. This includes the design of transcriptionaloscillators,59 the transcription template was activated by a DNA-fuel, and theactive DNA template was deactivated by displacement with an RNA anti-fuelthat was degraded by RNase to reactivate the fuel strand. The secondapproach60 employed a reaction module consistingof a transcription template that was triggered by the ribonucleosidetriphosphate (NTP) fuels to yield a RNA product that resulted in thestructural displacement of the reaction module that led to the formationof a catalytically active DNAzyme and an intermediate RNA/DNA duplexthat was digested by RNase, leading to the transient dissipative formationand depletion of the DNAzyme intermediate product. The third method61 involved the design of a reaction module consistingof two transcription templates and an inhibited aptamer/T7 RNA polymerase(RNAP) complex. The fuel-driven separation of the aptamer/RNAP complexactivated the RNAP/NTPs operation of the two transcription templates,where one led to the target RNA and the second template transcribedthe inhibiting T7 RNAP aptamer, leading to the temporal transientinhibition of the central transcription machinery. These methods tostimulate the transient operation of transcription machineries suffer,however, from basic limitations, preventing the possibility to developcomplex transcription pathways. While the RNase-based systems consumethe RNA products, thus limiting their subsequent uses, the transcription-inducedgeneration of an inhibiting RNAP aptamer introduces a competitivepathway that utilizes most of the NTPs fuels, thus prohibiting theeconomic use of the fuels for complex transcription processes.In fact, in order to enhance the complexity of biomimetic transcriptionmachineries , it is essential todevelop dynamically controlled, transiently operating transcriptiontemplates. Three general methods to engineer temporally operatingtranscription templates were reported. By one methodHere we introduce three alternative strategies to promote transienttranscription machineries overcoming the outlined limitations associatedwith the RNase systems or the RNAP inhibiting concept. These includethe application of a DNAzyme-functionalized transcription template,a strand displacement dynamic inhibition of the transcription template,and a nickase-controlled transient operation of the transcriptiontemplate. Besides the introduction of alternative mechanisms to operatetransient transcription machineries, the systems are employed to guidegated transcription processes. Moreover, we present the possible useof transiently operating transcription machineries to assemble gated,transiently operating catalytic systems (DNAzymes).2+-ion-dependentDNAzyme and the transient transcription of the Malachite Green (MG)aptamer or the ((5Z)-5-[methylene]-3,5-dihydro-2,3-dimethyl-4H-imidazol-4-one) (DFHBI) broccoli aptamer are presented.The reaction module consists of the inactive T1/A1 template, where T1 includes the sequence-specific Pb2+-ion binding loop and the respective substrate-binding armsand the strand A1 is engineered to yield upon transcriptionthe MG-RNA aptamer. T7 RNAP, MG, the NTPs mixture, and Pb2+ ions are included in the reaction module. Under these conditions,the transcription template is incomplete and the transcription processis blocked. Subjecting the reaction module to the ribonucleobase-modifiedstrand Lr acting as a promoter complementing the transcriptiontemplate, yet engineered to act as substrate of the DNAzyme, resultsin the activation of the transcription machinery and the expressionof the MG-RNA aptamer. The binding of the promoter to the transcriptionmachinery and the triggered formation of the MG aptamer, however,lead to the concomitant Pb2+-ion-dependent DNAzyme-inducedcleavage of Lr, resulting in the degradation of the promoterunit to waste products and their separation from the transcriptiontemplate, leading to the recovery of the rest inactive reaction moduleand to the temporal, transient transcription of the MG aptamer. Thetemporal synthesis of the MG-RNA aptamer is, then, followed by thetemporal fluorescence changes of the MG aptamer complex. ex = 635 nm and λem = 665 nm) upon subjectingthe reaction module to different concentrations of Lr.As the concentration of Lr increases, the time-dependentfluorescence changes are intensified, consistent with increased concentrationof the active transcription template synthesizing the MG-RNA aptamer.The time-dependent fluorescence changes decrease with time and reachsaturation values consistent with the DNAzyme-stimulated blockageof the transcription machinery and transient recovery of the inactivereaction module. The dynamic temporal fluorescence changes of the MG aptamer complexgenerated by the reaction module were then translated into catalyticrates forming the MG-RNA aptamer  By re-addition of the triggeringribonucleobase-modified promoter Lr, the transient, temporalreactivation of the transcription machinery was demonstrated, 2+-ion-dependentDNAzyme-controlled transcription of the MG aptamer, see Figure S7 and the accompanying discussion.)The first system to assemble a transient dissipative transcriptionmachinery is displayed in 2+-ion-dependent DNAyzme-guidedtransient transcription machinery synthesizingthe DFHBI-RNA aptamer. In this case, a reaction module consistingof transcription template T2/A2, T7 RNAP, DFHBI,and NTPs was assembled, Figure S8. Thestrand A2 included an engineered sequence for transcriptionof the DFHBI-RNA aptamer. The reaction module was triggered with theribonucleobase-modified promoter Lr, and the temporal transienttranscription of the DFHBI-RNA aptamer was followed by time-dependentfluorescence changes of the DFHBI aptamer complex (λex = 470 nm and λem = 500 nm). r, and r. Slightly slower DNAzyme-drivendepletion rates of this transcription machinery, as compared to theMG aptamer transcription machinery are observed (the recovery of theinactive reaction module and blockage of the transcription machineriesare observed after ca. 75 min). As before, by re-addition of the triggeringstrand Lr, the transient, DNAzyme-guided transcriptionmachinery was reactivated, The same concept wasapplied to design an alternate Pb1/A1 and T2/A2 that included the Pb2+-ion-dependent DNAzyme loops as functional units. T7 RNAP,NTPs, MG, DFHBI, and Pb2+ ions were included in the reactionmodule. In the presence of the common triggering promoter strand Lr, the Pb2+-ion-dependent DNAzyme-controlled transienttranscription templates T1/A1 and T2/A2 are activated, leading to the concomitant formationof the MG and DFHBI aptamers reflected in the temporal fluorescenceintensities of the MG aptamer and DFHBI aptamer complexes and theaccompanying transient catalytic rates generating the aptamers, 1, resultsin the hybridization of the blocker 1 with the DNAzymetether unit associated with T1. The tight duplex betweenblocker 1 and T1 prohibits binding of thepromoter unit, Lr, to T1. Subjecting the reactionmodule, consisting of the 1-blocked configuration, StateII, to the ribonucleobase-modified strand Lr activatesthe gated transcription of template T2/A2 whilethe operation of template T1/A1 is switchedoff. This is reflected by the Pb2+-ion-dependent DNAzymeguided transient fluorescence changes of the DFHBI aptamer complex, 2, results in, however, the selective blockingof template T2/A2, State III, leading to thegated transcription of the MG-RNA aptamer. This is reflected withthe selective, switched-on, temporal fluorescence changes of the MGaptamer complex, The DNAzyme-guided transient operationof the transcription machinerieswas then applied to assemble a gated transcription reaction module, 3/A1 and T4/A3, T7 RNAP, NTPs, and the MG ligand. Subjecting the reaction moduleto the fuel strand F activates the two transcription machineries.While activation of T3/A1 leads to the transcriptionof the MG-RNA aptamer as product, activation of the template T4/A3 leads to the transcription of the RNA productF′ that acts as an anti-fuel strand displacing the activatorstrand F. That is, formation of F/F′ duplex regenerates therest reaction module, leading to the transient operation of the MGaptamer transcription machinery. Supporting Information and accompanyingdiscussion.) Figure S11. For a control experimentof strand-displacement-stimulated transcription machinery suggestingthe inhibition of leakage of the transcription machinery in the absenceof the fuel strand F, see Figure S12. Foradditional experiments probing the effect of different concentrationsof the NTPs and variable concentrations of the template T4/A3, see Figures S13 and S14. For further electrophoretic experiments supporting the transienttemporal operation of the dual transcription machineries, see Figure S15 and accompanying discussion. For additionalcontrol experiments employing a triggering strand, Fc, not being displacedby the transcribed F′, and an experiment probing the stabilityof template T4/A3 in the presence of fuel strandF, see the Supporting Information, FiguresS16 and S17 and accompanying discussions.) Figures S18–S20.)The second approach to trigger the transcription apparatusandto stimulate the gated operation of the transcription machineriesis displayed in 3/A1, T4/A3, and T5/A2, the T7 RNAP, NTPs, and the ligands MG and DFHBI, State I. Subjectingthe reaction module in State I to the fuel strand F, activates allthree templates. The activation of the two templates T3/A1 and T5/A2 leads to the transcriptionof the MG-RNA aptamer and the DFHBI aptamer, followed by time-dependentfluorescence changes of the MG aptamer complex and the DFHBI aptamercomplex. The concomitant activation of the template T4/A3 leads to the transcription of the strand F′ that displacesthe activating strand F associated with T3, T4, and T5, resulting in the depletion of the active templatesand to the transient synthesis of the MG aptamer and DFHBI aptamerproducts. 3 leads to thehybridization of the blocker with the template T3/A1. Under these conditions, in the presence of the fuel strandF, only the transcription templates T5/A2 andT4/A3 are activated, resulting in the transienttranscription of the DFHBI aptamer and the blockage of the MG aptamertranscription template, T3/A1, State II. PanelsV and VII show the temporal fluorescence changes associated with theformation of the MG aptamer and the DFHBI aptamer by the reactionmodule in State II. While the formation of the MG aptamer is blocked,effective temporal fluorescence changes of the DFHBI aptamer complexthat levels off after ca. 15 min are observed. Similarly, treatmentof the reaction module in State I with the blocker unit 4 yields the reaction module in State III, where template T5/A2 is blocked, and the gated triggered activation ofthe template T3/A1 and T4/A3 by the fuel strand F proceeds. Panels IX and XI depict the experimentalresults demonstrating the temporal increase and leveling off of thefluorescence intensities of the MG aptamer complex, while the fluorescenceof the DFHBI aptamer complex is blocked. Similarly, Panels X and XIIshow the gated transient time-dependent rates of formation of theMG or DFHBI aptamer by State III. Evidently, the integration of twotranscription machineries activated by a common fuel activating strandand an auxiliary transcription machinery activated by the fuel strandto yield an anti-fuel strand displacing the activating strands, associatedwith all transcription templates, leads to the competitive dissipationof all transcription machineries. In the presence of appropriate blockerunits, gated operation of the transcription templates is demonstrated.6/A4, the mixture NTPs, the T7 RNAP, and the nicking enzyme Nt.BbvCI.Subjecting the reaction module to the fuel strand L1, resultsin its hybridization with a part of the free single-strand tetherT6. Hybridization of L1 with the template completesthe promoter-activated machinery, resulting in transcription of theMG-RNA aptamer. The strand L1 was encoded, however, toinclude the sequence-specific domain to be nicked by Nt.BbvCI. Cleavageof L1 leads to “waste” fragments, being separatedfrom the transcription template, resulting in the blockage of thetranscription process and the recovery of the inactive reaction module.This dynamic set of reactions leads to the transient temporal formationof the MG-RNA aptamer, a process that is followed by the transientevolution of the fluorescent MG aptamer complex. 1. The fluorescence intensitiesare higher as the concentrations of L1 increase. Theseresults are consistent with the enrichment of the active transcriptiontemplate upon elevating the concentration of L1 and thetemporal blockage of the transcription machinery by the nickase-stimulateddepletion of the active transcription template, leading to a constantaccumulation of MG-RNA aptamer.  The temporal fluorescence changes of the MG aptamer depictedin 1, and these are displayed in Figure S23. For a control experiment of nickase-driven transcription machinerydemonstrating the inhibited lack of leakage of the transcription machinery,in the absence of the fuel strand L1, see Figure S24. For additional experiments probing the nickase-guidedtransient operation of the transcription machinery in the presenceof variable concentrations of NTPs and nickase, see Figures S25 and S26.) The reaction module can be reactivatedby re-addition of the fuel strand L1, 7/A5 and the nickaseNb.BtsI . 2. As theconcentration of L2 increases, the fluorescence changesin the DFHBI aptamer are higher. 2 (time marked with arrow).A third mechanism to regulate the operation of transcription templatesusing a fuel-strand as activator and a nickase as catalyst dissipatingthe active structure of the template is introduced to stimulate transienttranscription machineries and gated, transient transcription machineries. 6/A4 and T7/A5, two sequence-specificnickases, Nt.BbvCI and Nb.BtsI, T7 RNAP, the NTPs, and the ligandsMG and DFHBI. Subjecting the reaction module to the triggering strandsL1 and L2 activates the two templates, resultingin the operation of the two transcription machineries and the formationof the MG-RNA aptamer and DFHBI-RNA aptamer. The concomitant nickingof the strands L1 and L2 by the Nt.BbvCI andNb.BtsI nickases degrades the respective templates, leading to thetransient transcription of the two RNA aptamers and the recovery ofthe parent inactive reaction module. 5 blocks the template T6/A4, prohibitingthe activation of the template T6/A4 by L1, State II. As a result, in the presence of the triggers L1 and L2, the gated activation of template T7/A5 leads to the selective transcription of theDFHBI-RNA only. 6 yields State III, where temporal T7/A5 isblocked. This leads, in the presence of L1 and L2, to the selective operation of the template T6/A4, resulting in the temporal, transient transcription of theMG-RNA aptamer, Moreover, as before, the nickase-stimulated transient transcriptionmachinery was further engineered to drive gated transient machineriesin the presence of two different nickases and appropriate blockerunits, as shown in 2+-ion-dependentDNAzymes. The reaction module, State I, consists of two inactive transcriptiontemplates T8/A6 and T9/A7, the T7 RNAP, NTPs, and the two nicking enzymes, Nt.BbvCI and Nb.BtsI.In the presence of the triggering strands L1 and L2, the two transcription templates are activated to yield thetranscription products R1 and R2. The concomitantnicking of the triggering strands L1 and L2,associated with the templates by the two nickases, Nt.BbvCI and Nb.BtsI,depletes the active transcription templates and drives the recoveryof the parent, rest reaction module, leading to the transient andtemporal transcription of the products R1 and R2. The latter products act, however, as precursors for the transientoperation of two different Mg2+-ion-dependent DNAzymes.Extrusion of samples at time intervals of the operating temporal,transient transcription of the products R1 and R2 into a mixture consisting of the duplexes P1/Q1 and P2/Q2 and the DNAzyme subunits X, Y andZ, U and the respective fluorophore/quencher- and ribonucleobase-modifiedDNAzyme substrates, S1 and S2, results in asequence of processes generating the temporal formation of two Mg2+-ion-dependent DNAzymes, DNAzyme 1 and DNAzyme 2, and to the temporal, transient cleavage of the substratesS1 and S2. The transcription products R1 and R2 displace the duplexes P1/Q1 and P2/Q2 to yield the duplexes R1/Q1 and R2/Q2, and the releasedstrands P1 and P2 are pre-engineered to assemblethe DNAzyme 1 and DNAzyme 2. The two assembledDNAzymes cleave the respective fluorophore/quencher-labeled substrates(FAM/IBRQ for S1 and Cy5/BHQ2 for S2), and theresulting temporal fluorescence changes probe the temporal activitiesof DNAzyme 1 and DNAzyme 2. Note that thetwo Mg2+-ion-dependent DNAzymes 1 and 2 differ in the sequence of the binding arms, leading to thespecific cleavage of the two different substrates S1, S2. Moreover, because the transcription machineries guide thetemporal transient formation of R1 and R2, thedynamic features of the transcribed products are translated into dynamiccatalytic features of the resulting DNAzymes. That is, the DNAzymesreveal transient catalytic activities. 1 and S2, respectively.The insets in Panels I and II present the derived curves of the transientoperation of catalytic rates of the DNAzymes, imaging the parent transienttranscription rates generating R1 and R2. Subjectingthe reaction module in State I to the blocker unit 6 inhibitsthe transcription template T9/A7, leading tothe gated operation of the template T8/A6, StateII. This leads to the transient formation of R1 and thesecondary gated operation of DNAzyme 1, cleaving S1. Panels III and IV show the temporal catalytic rates of theDNAzymes upon operating the 6-inhibited reaction modulein State II, demonstrating the effective temporal catalytic ratesof DNAzyme 1 and the blocked catalysis of DNAzyme 2. Similarly, treatment of the reaction module in State Iwith the blocker unit 5 leads to the gated inhibitionof the template T8/A6, State III. Under theseconditions, template T9/A7 is activated towardthe transient transcription of R2 and the subsequent R2-guided operation of DNAzyme 2. Panels V andVI show the temporal catalytic rates upon the 5-inhibitedoperation of the gated reaction module in State III. While DNAzyme 2 reveals temporal cleavage of the substrate S2 and transient catalytic rates, guided by R2, DNAzyme 1 is inhibited.Following the development of three alternativemethods drivingtransient transcription machineries and gated transient transcriptionsystems, we aimed to demonstrate the possible utility of these dynamictransient transcription networks, particularly toward the assemblyof transcription-guided catalytic processes. This is exemplified in Figures S31–S33, with the parallel operation of two transient transcription machineriesusing the DNAzyme-modulated, strand-displacement-modulated, and nickase-driventranscription machineries transcribing the MG-RNA aptamer and theDFHBI-RNA aptamer, respectively.The study introduced three alternativemethods to trigger transientmachineries. The results raise, however, fundamental questions relatedto possible advantages/disadvantages associated with the differentmethods and possible benefits of having three alternative triggeredtransient transcription machineries. Upon comparing the nickase-andDNAzyme-modulated transient machineries to the strand-displacement-modulatedmachinery, we may realize that the latter is less economic due tothe parallel consumption of the NTPs bases. Moreover, the nickase-modulatedtriggered transcription machinery requires an additional enzyme tothe T7 RNAP, adding disadvantages associated with the stability ofthe nickase and eventually the need to adjust auxiliary system conditionssuch as temperature or buffer composition. Furthermore, the availabilityof three alternative triggering modes to stimulate the transcriptionmachineries introduces further possibilities to enhance the complexityof the systems by integrating several of the transcription machinerymodes into a single system operating in parallel. Under such conditions,the integration of different transcription templates, different respectivetriggering fuels, and engineering of the templates to yield differentRNA output products, while ensuring lack of destructive inhibitionor crosstalks between the system constituents, is essential. Thisis exemplified in Thepresent study has introduced three alternate methods to operatetransient transcription machineries and gated transient transcriptionmachineries—the design of reaction modules that include thedynamic DNAzyme-, strand-displacement-, or nickase-stimulated dissipationof the active transcription templates, leading to the transient activitiesof the systems. All three methods were used to operate the gated transientsynthesis of two different RNA aptamers by integrating two alternativetranscription templates into the reaction modules and dictating theoperation of the desired machinery by applying appropriate blockers.The significance of these methods rests, however, on diversity andenhanced complexity capacities to operate multimode transcriptionmachineries synthesizing selective mixtures of RNAs. The reactionmodules could include any number of transcription machineries andmodulating blocker units that control the number and composition ofevolved RNAs using appropriate regulators. Furthermore, by the integrationmore than one regulator, e.g., different strand displacers or differentnickases, the complexities of the systems might be even further enhanced.58 or liposomes.57 Theintegration of transiently modulated transcription machineries intosuch cell-mimicking microenvironments might be an important step infuture “protocell” design.56At present, the different transiently modulated transcription circuitsare operating in homogeneous buffer solutions. Recent efforts aredirected, however, to integrate triggered transcription machineriesinto cell-like containments, such as hydrogel microcapsulesOligonucleotideswere purchased from Integrated DNA Technologiesand Sigma-Aldrich. The sequences of the oligonucleotides are listedin Supporting Information.Detailedprocedures to prepare and perform the different transienttranscription systems and to characterize the systems are presentedin the


Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax (Ubx) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as an lncRNA promoter. To activate Ubx expression in PS6/A1, enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that the expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to read-through as the HS2 promoter must be directed toward HS1 to disrupt blocking.Though long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C)  HOTT1P, encode RNA sequences that recruit chromatin modifiers that help promote gene expression , not mRNAs. Their wide distribution in the genome and the fact that their expression is often coordinated with genes in the immediate vicinity have led to the idea that they have functions in gene regulation and chromosome organization . Unlike pression . In othepression . For exaell fate . TranscrUltrabithorax (Ubx), in the Drosophila bithorax complex (BX-C). Ubx, together with abdominal-A (abd-a) and Abdominal-B (Abd-B), are responsible for specifying the identity of the nine parasegments (PS)/segments that form the posterior two-thirds of the fly (Ubx is responsible for specifying parasegments PS5 (adult cuticle segment T3) and PS6 (adult cuticle segment A1). Its expression in these two parasegments is controlled by the abx/bx and bxd/pbx regulatory domains, respectively. abd-A expression in PS7-PS9 is directed by the iab-2–iab-4 regulatory domains, while Abd-B expression in PS10-14 is regulated by the iab-5–iab-9 regulatory domains. Each regulatory domain has an initiation element, a set of tissue-specific enhancers, and Polycomb Response Elements (PREs)  only the abx/bx domain is activated  and it is responsible for regulating Ubx expression in this parasegment. Both abx/bx and bxd/pbx are turned on in PS6(A1); however, Ubx expression and parasegment identity depend on the bxd/pbx domain. Once the activity state of the domain is set in early embryos, it is remembered during the remainder of development by the action of Polycomb Group proteins (PcG: off) and Trithorax group proteins (Trx: on). Each domain also contains tissue- and stage-specific enhancers that drive the expression of its target homeotic gene in a pattern appropriate for proper development of the parasegment it specifies.In the studies reported here, we have investigated the role of lncRNAs in regulating the chromatin organization and expression of the homeotic gene,  the fly . Parasegs (PREs) . Early iAbd-B region of the complex. Mutations that inactivate Abd-B boundaries cause a gain-of-function (GOF) transformation in segment identity. For example, the Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that are responsible for specifying PS11(A6) and PS12 (A7) identity, respectively  and iab-7 instead of iab-6 drives Abd-B expression in this parasegment as well as in PS12(A7). This results in the duplication of the PS12/A7 segment. However, blocking crosstalk between adjacent regulatory domains is not the only function of BX-C boundaries. The three homeotic genes and their associated regulatory domains are arranged so that all but three of the domains are separated from their targets by one or more intervening boundaries. For this reason, all but two (Fub and Mcp) of the internal BX-C boundaries must also have bypass activity ) or by BX-C boundaries that lack bypass activity (Mcp), these boundaries block crosstalk but do not support bypass  phenotype.In order to properly specify parasegment identity, the nine BX-C regulatory domains must be functionally autonomous. Autonomy is conferred by boundary elements (insulators) that flank each domain . The rolectively . When Faactivity . For exat bypass . As a coUbx domain is delimited and subdivided by three boundaries called Fub-2, Fub-1, and Fub. Fub-2 and Fub-1 flank abx/bx domain, whereas bxd/pbx is flanked by Fub-1 and Fub , while it is turned on in A1/PS6 and more posterior parasegments. The bxd/pbx domain is bracketed by the Fub boundary on the centromere distal side and Fub-1 on the centromere proximal side. The chromosomal architectural proteins CTCF, Pita, and Su(Hw) are associated with Fub in ChIP experiments, and like boundaries the Abd-B region of BX-C Fub deletions result in a GOF transformation of PS6(A1) into PS7(A2). The Fub-1 boundary has only been defined molecularly. ATAC-seq mapping in nuclear cycle 14 embryos indicates that this boundary includes two chromatin-specific transposase hypersensitive regions: HS1 ~200 bp and HS2 ~300 bp  and more posterior parasegements, the bxd/pbx domain also controls the expression of two lncRNAs, bxd and HS2Fub-1 HS2, Fub-1. Pease et al. found that the bxd lncRNA initiates from a promoter close to the pbx enhancer and extends into the region located in between Fub-1 and the Ubx promoter  and more posterior parasegments. This lncRNA is encoded by sequences located between Ubx and Fub-1, including sequences in Fub-1 HS1, and based on the in situ experiments of Pease et al., it likely originates from HS2Fub-1 HS2. Fub-1 shares exon sequences with the bxd lncRNA. However, the HS2Fub-1 lncRNA is detected in PS6/A1 and more posterior segments after the bxd lncRNA disappears.Besides directing the expression of promoter . The bxdFub-1 hypersensitive sites are evolutionarily conserved. As shown in HS1 and HS2 are detected not only in other members of the Sophophora subgenus, but also in Drosophila virilis, a distantly related member of the Drosophila subgenus. The conserved blocks in HS1 include the dCTCF recognition site, while in HS2 the conserved blocks include several GAGA motifs that correspond to binding sites for the GAGA factor (GAF) as well as the predicted transcription start sites for the HS2Fub-1 lncRNA.As has been reported for the regulatory elements associated with other lncRNAs, the Fub-1 marks the border of a Polycomb histone H3K27me3 domain in PS5 cells would predict that Fub-1 delimits the endpoint of two adjacent TADs. The centromere distal TAD would encompass the bxd/pbx regulatory domain and end at Fub boundary that is located downstream of the abd-A transcription unit  are encompassed in a large TAD that extends from Fub-2 on the centromere proximal side of Ubx to Fub on the distal side. Within this large TAD, there is an ~50 kb sub-TAD that spans bxd/pbx and is delimited by Fub-1 on the left and Fub on the right . The endpoint for one of these HDIC domains maps to Fub-1, while the other endpoint maps to the bxd PRE. There is a second HDIC domain linking Fub-1 to sequences just upstream of the Ubx promoter. The Ubx promoter is also linked to the bxd PRE by a lower density of internal contacts domain (LDIC) that is marked at the apex by an interaction dot (*). The bxd PRE also forms an interaction dot (>) with the bx PRE as does the Ubx promoter (x). Interestingly PREs in other developmental loci have recently been shown to interact with each other and also function as ‘tethering’ elements helping to link distant enhancers to their target genes  that retains an attP site for boundary replacement experiments  toward A1 (PS6). Unexpectedly, however, attPF1 flies do not show any evidence of a GOF transformation and their morphology is indistinguishable from wild type (WT)  .Fub-1 and block crosstalk between abx/bx and bxd/pbx. Fub-1 to Fub that encompassed the bxd/pbx regulatory domain disappears in the Fub-1 deletion. However, a new sub-TAD that includes Fub does not appear to be formed. Instead, the element located just upstream of the Ubx promoter forms a sub-TAD domain with the bxd PRE (see interaction dot in abx/bx and bxd/pbx when the activity state of these domains in T3 (PS5) is set during early embryogenesis.A plausible explanation for this result is that there is a nearby element that can substitute for Fub-1 defines one endpoint of a TAD and marks the border for the PcG H3K27me3 histone mark in PS5 nuclei, the finding that Fub-1 deletions have no phenotype raises the possibility that it is actually ‘within’ the bxd/pbx regulatory domain. If this is the case, then reporters inserted between Fub-1 and the element just upstream of the Ubx promoter should respond to bxd/pbx enhancers, but be insulated from enhancers in the abx/bx regulatory domain. To test this possibility, we analyzed the pattern of expression of reporters inserted into the attPF1 site with and without the Fub-1 boundary.Although DsRed reporter used to mark the Fub-1 deletion in attPDsRed-F1. DsRed is expressed throughout the posterior ~2/3rds of Fub-1 deletion embryos, with an anterior limit corresponding to the anterior border of PS6/A1. Thus, as expected from the lack of phenotypic effects of the Fub-1 deletion, the reporter is insulated from the abx/bx domain, but is subject to regulation by the bxd/pbx domain. We next introduced the 1168 bp Fub-1 fragment together with a GFP reporter flanked by frt sites back into the attPDsRed-F1 platform , and this reporter is also ‘located’ within the bxd/pbx regulatory domain.We first examined the platform . In the ub-1-GFP . As showUbx promoter displayed two distinct patterns of expression. An enhancer trap located 13 bp upstream of the Ubx transcription start site had a PS5/T3 anterior border, indicating that it is regulated by the abx/bx domain. In contrast, an enhancer trap located 196 bp upstream of the start site had an anterior limit of expression shifted to PS6/A1  replacement platform  inserted in the same 5′→3′ orientation as the endogenous Fub-1 element. attP50F7 deletion. This result indicates that these two Fub-1 fragments do not prevent crosstalk between iab-6 and iab-7. The 248-HS2505HS1 replacement GOF phenotype is reflected in the pattern of Abd-B expression in the embryonic CNS that closely matches that of the starting attP50F7 deletion  and included all of the HS1 sequences plus the sequences located between HS1 and HS2. The other, a 248 bp fragment (R6.22: 3R: 16748578–16748825), contains only the HS1 region. These fragments were inserted in both the forward and reverse orientations. We also tested a 505 bp fragment spanning the HS2 (R6.22: 3R: 16748826–16749330) in both the forward and reverse orientations  by Lefevre et al. showed that LPS treatment activated the transcription of an lncRNA through a CTCF boundary  was inserted in the forward orientation so that the HS2 element is ‘pointing’ toward the iab-6 domain  is sufficient to rescue the blocking activity of HS1. As observed for HS1 alone, 505R-HS1248R-CTCFx4HS2 males have a tergite and sternite whose morphology resembles that seen in A5. Likewise, the pattern of Abd-B expression in the CNS in 505R-HS1248R-CTCFx4HS2 is similar to that observed for 248HS  could disrupt or HS248 .Fub-1 likely functions as a boundary in its endogenous context; however, its boundary activity is segmentally regulated by transcriptional read-through from HS2. This model makes two predictions. First, the activity of the HS2 promoter should be controlled by the bxd/pbx regulatory domain. Second, when transcriptional read-through is prevented, Fub-1 blocking activity would be expected to interfere with Ubx regulation by the bxd/pbx regulatory domain. We have tested these predictions.Taken together, the findings in the previous sections suggest that bxd domain located just proximal to Fub-1  Fub-1.In order to test the first prediction, we used smFISH probes spanning a 1983 bp region in the to Fub-1 . As noteWT and attPF1 embryos as early as nuclear cycle 13. In WT, the smFISH probe is expected to hybridize to both HS2Fub-1 and bxd lncRNAs. In contrast, since the HS2Fub-1 promoter is deleted in attPF1 embryos, only bxd lncRNAs should be detected. As would be expected if both bxd and HS2Fub-1 lncRNAs are expressed at this stage in WT embryos, the intensity of the smFISH signal is higher in WT compared to attPF1. During gastrulation, the broad band seen at nuclear cycle 13 resolves into a series of stripes of near equal width and intensity extending toward the posterior of the embryo  and postbithorax (pbx), respectively  anterior to PS6/A1 at the blastoderm stage and is repressed during the remainder of development by PcG-dependent silencing. In PS6/A1 (and more posterior segments), the bxd/pbx regulatory domain is set in the on state, activating the stage and tissue-specific enhancers in this domain. These stage- and tissue-specific enhancers activate the HS2 promoter in PS6/A1 and more posterior parasegments and transcription from this promoter through HS1 disrupts Fub-1 boundary function  element are able to block crosstalk between iab-6 and iab-7 and both replacements exhibit the same GOF transformations as the starting attPF7 replacement platform. In the HS1+HS2 Fab-7 replacement experiments, the defects in Fub-1 blocking activity arise from transcriptional read-through from the HS2 promoter. This conclusion is supported by two lines of evidence. First, introducing the SV40 transcription termination polyadenylation element in between HS2 and HS1 partially rescues the blocking defect. Rescuing activity is not due to the increased distance between HS2 and HS1 as a SV40 element with mutations in the polyadenylation signal significantly compromising its rescuing activity. In addition, the SV40 termination element must be located between HS1 and HS2 as rescue is not observed when it is placed downstream of HS1. Second, when HS2 is inverted so that transcription proceeds away from HS1, boundary function is also restored.Several lines of evidence are consistent with this mechanism. When Fub-1 boundary activity in its endogenous context; however, in this case boundary activity is segmentally regulated. In PS5 and more anterior parasegments, the bxd/pbx regulatory domain is maintained in an ‘off’ state by a PcG-based mechanism. As a consequence, the Fub-1 HS2 promoter is inactive  bxd/pbx. Fub-1 also corresponds to a boundary for two smaller HDIC domains, one with a second endpoint close to the Ubx promoter and another with a second endpoint close to the bxd PRE. In the Fub-1 deletion, the proximal endpoint of the subTAD formed by Fub-1 and bxd PRE shifts to the Ubx promoter region, while the two HDIC domains collapse into one. If the function of HS2 is to inactivate HS1 in the posterior 2/3rds of the embryo, one would expect that this would disrupt the Fub-1 ←→ Fub TAD, much like the Fub-1 deletion. However, we observe a Fub-1 ←→ Fub TAD. Since the HS2 promoter should not be active in cells anterior to PS6/A1, it is possible that these cells are responsible for generating the Fub-1 ←→ Fub TAD seen in the Micro-C experiments. Alternatively (or in addition), the disruption in HS1 function might only occur during a burst of transcription (HS1 blocking activity is directly coupled to the act of transcription (as appears to be the case), then there may only be small windows of time in which the activity of the Fub-1 boundary is compromised and bxd/pbx enhancers can activate the Ubx promoter. In this case, the Fub-1 insulator would flip back and forth from an inactive and active state (WT and 540R-HS1248RHS2). Potentially consistent with a model along these lines, in stage 14 embryos, only a subset of cells in PS6 and more posterior parasegments have transcripts complementary to HS2Fub-1 (Another question is raised by the Micro-C contact pattern in cription . Recent cription . The frecription . For othcription . If disrFub-1HS2 .HS2 transcription when the Fub-1 boundary is reversed (the 248-HS2505RHS1 replacement) so that HS1 is interposed between HS2 and the bxd/pbx domain. One would have expected that HS1 would block the bxd/pbx enhancers from activating HS2 transcription. In this case, boundary function would be retained, resulting in an LOF transformation of PS6/A1 into PS5/T3 because it would prevent bxd/pbx from regulating Ubx. One likely possibility is that the putative enhancers in the region between the Ubx promoter and Fub-1  cells and this would inactivate the blocking activity of HS1 in cell in this parasegment, giving a GOF transformation of PS11/A6. The unexpected result is that a GOF transformation of PS11/A6 into PS12/A7 is also observed when the Fub-1 replacement is inserted so that HS2 is next to iab-7. Since iab-7 is ‘off’ in PS11, this would imply that HS2 must be activated by the iab-6 regulatory domain. A similar scenario could be at play when Fub-1 is inserted in the opposite orientation in its endogenous location: HS1 blocks but does not completely eliminate interactions between the bxd/pbx enhancers and the HS2 promoter. This is probably not unexpected. While interposed boundaries significantly reduce enhancer:promoter contacts, they are not completely eliminated  so that the bxd/pbx domain can regulate Ubx expression. Interestingly, like cis-acting elements for many other lncRNAs, both of the Fub-1 boundary subelements are conserved in the Sophorphora subgenus  that is commonly encountered by a wide range of Drosophila species.Perhaps the most puzzling question is what function does subgenus . It is a+); TM6, Tb, Hm/Sb, w[1]; TM6, Tb, Hm/Sb, P{w[+mC]=ovoFLP.R}M1A, w[*] (BDSC_8727), y[1] w[67c23] P{y[+mDint2]=Crey}1b; D[*]/TM3, Sb[1] (BDSC_851) was used for all transgenesis. y[1] M{GFP[E.3xP3]=vas-Cas9.RFP-}ZH-2A w[1118] (BDSC_55821) was used for CRISPR/Cas9 genome editing. y[1] w[67c23] P{y[+mDint2]=Crey}1b; D[*]/TM3, Sb[1] (BDSC_851) was used for Cre/loxP recombination to remove DsRed from CRISPR-generated DsRed insertions. w[1]; TM6, Tb, Hm/Sb, P{w[+mC]=ovoFLP.R}M1A, w[*] (BDSC_8727) was used for Flp/frt recombination to remove GFP from ΦC31-mediated insertions. 248-HS2505RHS1 stock is weak and was grown on rich medium described by The following stocks were used in these experiments: y[1] M{GFP[E.3xP3]=vas-Cas9.RFP-}ZH-2A w[1118] (BDSC_55821), w[1], ΦC31 , and TTGCGGCCGCATTTCTACGTTTATAAGCTGTTAATC and AAGCATGCCCACAATGGAGGCTGC for the distal arm (1002 bp fragment). Targets for Cas9 were selected using ‘CRISPR optimal target finder’—the program from O'Connor-Giles Laboratory. The following gRNA sequences, AGTCGATCGTAAACCGGAG and TTATAGACGTAGAAATGTA, were inserted into the pU6-BbsI-chiRNA vector (Addgene plasmid no. 45946). A mixture of the donor vector (500 ng/ml) and two gRNA vectors (100 ng/ml each) was injected into the embryos of y[1] M{GFP[E.3xP3]=vas-Cas9.RFP-}ZH-2A w[1118] (no. 55821 from the Bloomington Drosophila Stock Center). Injectees were grown to adulthood and crossed with w[1]; TM6/Sb line. Flies positive for dsRed signal in eyes were selected into a new separate line. The successful integration of the recombination plasmid was verified by PCR and corresponded to the removal of 1168 bp within the Fub-1 region .For generating double-stranded DNA donor template for homology-directed repair, we used pHD-DsRed vector (Addgene plasmid no. 51434). The final plasmid contains genetic elements in the following order: [bxd proximal arm]-[attP]-[loxP]-[3×P3-dsRed-SV40polyA]-[loxP]-[bxd distal arm] . HomologattPF1 replacement, the recombination plasmid was designed de novo and contains several genetic elements in the following order: [loxP]-[pl]-[frt]-[3P3-GFP]-[frt]-[attb] (DsRed marker gene and plasmid body. Two frt sites are used to excise GFP marker. DNA fragments used for the replacement experiments were generated by PCR amplification and verified by sequencing (presented in the Supplementary Methods).For the ]-[attb] . All eleThe strategy of the Fab-7 replacement lines creation is described in detail in 2O two times and heated again in dH2O for 1 hr. After that, the digested flies were washed three times with 70% ethanol and stored in 70% ethanol. The abdomen cuticles were cut off from the rest of the fly using a fine tweezer and a needle of an insulin syringe and put in a droplet of glycerol on a microscope slide. Then the abdomens were cut longitudinally on the mid-dorsal side through all of the tergites with the syringe. Then the cuticles were flattened with a coverslip. 248-HS2505RHS1 third instars were used for larval cuticle preparation. This stock is homozygous sterile and was maintained on TM6B balancer. The TM6B balancer carries the Tb1 dominant mutation, which results in tubby larvae and pupae. Larvae without tubby phenotype were selected for cuticle preparation. Larval cuticle was prepared as described by Three-day adult male flies were collected in 1.5 ml tubes and stored in 70% ethanol at least 1 d. Then ethanol was replaced with 10% KOH and flies were heated at 70°C for 1 hr. After heating, flies were washed with dHEmbryo immunostaining was performed as previously described . PrimarysmFISH was performed as previously described . Quasar Embryos were collected on yeasted apple juice plates in population cages. Plates were laid for 4 hr, incubated for 12 hr at 25℃, then subjected to fixation. Embryos were collected in nylon mesh sieves, dechorionated for 2 min in 3% sodium hypochlorite, rinsed with deionized water, and transferred to glass vials containing 5 ml PBST (0.1% Triton-X in PBS), 7.5 ml N-heptane, and 1.5 ml fresh 16% formaldehyde. Crosslinking was carried out at room temperature for exactly 15 min on an orbital shaker at 250 rpm, followed by addition of 3.7 ml 2 M Tris-HCl pH7.5 and shaking for 5 min to quench the reaction. Embryos were washed twice with 15 ml PBST and subjected to secondary crosslinking. Secondary crosslinking was done in 10 ml of freshly prepared 3 mM final DSG and ESG in PBST for 45 min at room temperature with passive mixing. Reaction was quenched by addition of 3.7 ml of 2 M Tris-HCl pH7.5 for 5 min, then washed twice with PBST. Embryos were snap-frozen and stored at –80℃ until library construction.Micro-C libraries were prepared as previously described  with theDrosophila melanogaster were aligned to a custom genome edited from the Berkeley Drosophila Genome Project (BDGP) Release 6 reference assembly  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Drosophila BX-C chromatin boundary Fub-1 is parasegmentally regulated by lncRNA read-through" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Ian Duncan (Reviewer #1).Thank you for submitting your article "The insulating activity of the First let me apologize for the long delay, which was due to administrative mixups, not science. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Given the central importance of the F1HS2 RNA to the story presented, it is disappointing that the structure and expression of this RNA are not well defined in the paper. In particular evidence of ncRNA transcript spanning HS1 in WT and absence in promoter inversion and premature termination strains would be important for the authors' model.2) Please provide detail on the larval and imaginal phenotypes of HS1248 HS2505R animals.3) Please address whether the direction of transcription of HS1 is important.Reviewer #1 (Recommendations for the authors):Page 5: The following sentences present what most readers will see as a contradiction:"However, when Fab-7 is replaced by generic fly boundaries (e.g. scs or su(Hw)) or by BX-C boundaries that lack bypass activity (Mcp) these boundaries block crosstalk but do not support bypass. As a consequence, Abd-B expression in PS11/A6 is driven by iab-5 not iab-6 and this results in a loss-of-function phenotype".How is it that iab-5 can drive Abd-B expression without bypassing the introduced generic boundaries? Others have attributed the ability of iab-5 to promote A5 identity in this situation to its ability to regulate abd-A.Page 5: "where multiple boundaries are located between the iab-5 and iab-6 regulatory domain" should read "where multiple boundaries are located between iab-5 and the Abd-B promoter" or something similar.Figure 1: Given the centrality of the bxd and F1HS2 RNAs to the story, it would help the reader if their locations were indicated in Figure 1.Page 7: It would help the novice reader if at the beginning of the Results or in the Introduction it were pointed out that the Ubx domain is delimited and subdivided by three boundaries called Fub-2, Fub-1, and Fub. Without this statement, some readers will, at least initially, confuse these boundaries with one another or think they are synonymous.Page 7 and General comment: Given the central importance of the F1HS2 RNA to the story presented, it is disappointing that the structure and expression of this RNA are not well defined in the paper. Although the characterization of F1HS2 is complicated by similar expression of the bxd RNA, the use of an existing strain in which the bxd promoter has been inverted could simplify this problem.Figure 2 legend: State here what F1attP is, what LDIC means, and that the data are from whole 12-18hr embryos.Page 10: "It would appear that this topological configuration is sufficient to block crosstalk between initiators in abx/bx and bxd/pbx when the activity state of these domains in T3 (PS5) is set during early embryogenesis." The logic here is difficult to follow. Either the argument should be expanded, or the sentence softened by substituting "It may be" for "It would appear that".Figure 4 and Supplementary Figure 4: In wild-type males, the A6 tergite differs from the A5 tergite in having large regions devoid of trichomes. In Supplementary Figure 4 it looks to me that genotypes in which HS1 elements without HS2 are inserted into F7attP50 all show regions in A6 that are devoid of trichomes, indicating some degree of A6 identity. However, the images are not entirely clear. It is very important to resolve whether patches devoid of trichomes are indeed present in the A6 tergite, because, if so, these patches would indicate that iab-6 is able to bypass the HS1 insulator to some extent. These same HS1 insertions into F7attP50 present a second problem for the reader: A5 and A6 are pigmented, indicating that iab-5 is also able to bypass the HS1 insulator. In the absence of clarification here, many readers will be mystified and discouraged from trying to make sense of what is a rather complicated paper.Figure 5: Contrary to the text, in part C, ventral nerve cord expression of HS2505R-HS1248R-SV40 and HS2505R-SV40-HS1248R look very similar to one another and to HS2505R-HS1248R. In addition, Abd-B expression in the ventral nerve cord should be shown for the HS2505R-mSV40-HS1248R genotype.In part E, the right-hand genotype I believe should be HS2505R-HS1248R.Page 20: Why reference Figure 2B here? Shouldn't this be 5E? The proper reference for the phenotypes of bxd and pbx mutations is definitely not Bender et al. – it should be one of the older Ed Lewis references!!!Page 23: "activate Ubx expression in PS5/A1" should be "activate expression in PS6/A1". Also "is abrogated in PS6/A1 and more anterior parasegments/segments" should be "abrogated in PS6/A1 and more posterior parasegments/segments".Page 26 top: "In the Fub-1 deletion, the proximal endpoint of the bxd/pbx TAD shifts to the Ubx promoter region". I don't see this shift in Figure 2 or in the schematic in Figure 7. Explain what is meant here or delete. In addition, "There could be several reasons for this discrepancy" is quite unclear. What discrepancy? Is it that one might expect the WT and F1attP data to look the same? The writing badly needs clarification here.Page 27: "In this case, iab-6 would drive HS2 expression in PS11(A7) cells" should be "In this case, iab-6 would drive HS2 expression in PS11(A6) cells".Reviewer #2 (Recommendations for the authors):Overall, the work was well planned and conducted, and the results are clearly presented and interpreted. As mentioned in the public review, my main question is how general and significant the mechanism is in insulator function and animal development. Below are specific points.1. Active transcription and chromatin boundaries are often found in close proximity. Can the authors check their micro-C and available PolII ChIP data from wt embryos to globally assess other known/novel boundary elements that show similar organization: an insulator (based on the presence of CTCF or other insulator protein binding sites) is near an active promoter that can transcribe through the insulator. In this way, can they predict other loci that may use a similar strategy in insulator regulation?2. In Figures 4 and 5, the authors nicely showed that depleting HS2, adding the SV40 terminator, and changing the direction of the promoter all restore the insulator activity of HS1. Although these manipulations are supposed to remove or disrupt the transcriptional read-through of the non-coding transcript, the authors should validate the assumption. The authors can perform PolII ChIP-qPCR on some of the transgenes to directly show that the read-through is gone, for example in the HS2505R-SV40-HS1248R and the HS1248-HS2505R animals (and HS2505R- HS1248R) as a control.3. Many promoters  are divergent, which means producing transcripts in both directions. However, the directionality of the HS2 promoter seems very strong, and maintaining such directionality is important for the function of HS2. Can the authors comment on what may ensure the directionality of the HS2 promoter?4. There are a few typos in the text, and some labeling mistakes for the figures . Essential revisions:1) Given the central importance of the F1HS2 RNA to the story presented, it is disappointing that the structure and expression of this RNA are not well defined in the paper. In particular evidence of ncRNA transcript spanning HS1 in WT and absence in promoter inversion and premature termination strains would be important for the authors' model.bxd and F1H2 transcripts are expressed in WT at the blastoderm stage. As the embryo develops, the bxd transcript disappears while the F1H2 transcript is still detected. For the anti-sense transcripts—we didn’t detect them in WT, but they were observed in both of inverted replacements.We have analyzed the “sense” lncRNA transcripts in WT and the Fub-1 deletion and anti-sense transcripts in WT, and the two HS2 promoter inversions. From a comparison of the smFISH hybridization pattern in WT and the Fub-1 deletion it is clear that both the 2) Please provide detail on the larval and imaginal phenotypes of HS1248 HS2505R animals.Photos showing larval phenotype are provided.3) Please address whether the direction of transcription of HS1 is important.Fub-1 with two other BX-C boundaries, Mcp and Fab-8. Both block bxd/pbx from activating Ubx expression. However, when a P-element promoter is introduced “upstream” of Mcp or Fab-8 (so it is pointing towards the boundaries and the bxd/pbx domain) it rescues the LOF phenotype. This would argue that the readthrough rather a particular feature of readthrough transcript is responsible for disrupting boundary activity.We didn’t do an experiment testing whether interruption of boundary activity required a “sense” transcript. However, some other experiments suggest that the transcript itself is unlikely to be relevant. We replaced Reviewer #1 (Recommendations for the authors):Page 5: The following sentences present what most readers will see as a contradiction: "However, when Fab-7 is replaced by generic fly boundaries (e.g. scs or su(Hw)) or by BX-C boundaries that lack bypass activity (Mcp) these boundaries block crosstalk but do not support bypass. As a consequence, Abd-B expression in PS11/A6 is driven by iab-5 not iab-6 and this results in a loss-of-function phenotype". How is it that iab-5 can drive Abd-B expression without bypassing the introduced generic boundaries? Others have attributed the ability of iab-5 to promote A5 identity in this situation to its ability to regulate abd-A.iab-5 generates an A5 segment via activation of abd-A. For example, other boundaries (e.g. Pitax5) are more effective in blocking iab-5 from activating Abd-B and give some LOF phenotypes in A5.Based on other experiments that we’ve done, it seems unlikely that Page 5: "where multiple boundaries are located between the iab-5 and iab-6 regulatory domain" should read "where multiple boundaries are located between iab-5 and the Abd-B promoter" or something similar.Typo – fixed.Figure 1: Given the centrality of the bxd and F1HS2 RNAs to the story, it would help the reader if their locations were indicated in Figure 1.Figure 1 is already very crowded. We added a schematic for bxd and Fub-1 ncRNAs in figure 7, right next to smFISH results.Page 7: It would help the novice reader if at the beginning of the Results or in the Introduction it were pointed out that the Ubx domain is delimited and subdivided by three boundaries called Fub-2, Fub-1, and Fub. Without this statement, some readers will, at least initially, confuse these boundaries with one another or think they are synonymous.This statement was added in introduction.Page 7 and General comment: Given the central importance of the F1HS2 RNA to the story presented, it is disappointing that the structure and expression of this RNA are not well defined in the paper. Although the characterization of F1HS2 is complicated by similar expression of the bxd RNA, the use of an existing strain in which the bxd promoter has been inverted could simplify this problem.Fub-1 deletion and the two HS2 inversion. Our analysis of the lncRNA transcripts in pre-cellular blastoderm embryos in WT and the Fub-1 using a probe adjacent to the Fub-1 deletion indicates that both bxd and F1HS2 transcripts are expressed at this stage. As the embryo develops the signal in the Fub-1 deletion disappears while it is readily detected in WT. By stage 11, only WT has transcripts complementary to this probe.We have analyzed lncRNA transcripts in WT, the Fub-1 deletion in WT and the inverse Fub-1 replacements. Antisense transcripts aren’t detected in WT but are in both of the inverse Fub-1 replacements.We also looked at antisense transcripts adjacent to the Figure 2 legend: State here what F1attP is, what LDIC means, and that the data are from whole 12-18hr embryos.We have rewritten Figure 2 legend.Page 10: "It would appear that this topological configuration is sufficient to block crosstalk between initiators in abx/bx and bxd/pbx when the activity state of these domains in T3 (PS5) is set during early embryogenesis." The logic here is difficult to follow. Either the argument should be expanded, or the sentence softened by substituting "It may be" for "It would appear that".The sentence is rewritten.Figure 4 and Supplementary Figure 4: In wild-type males, the A6 tergite differs from the A5 tergite in having large regions devoid of trichomes. In Supplementary Figure 4 it looks to me that genotypes in which HS1 elements without HS2 are inserted into F7attP50 all show regions in A6 that are devoid of trichomes, indicating some degree of A6 identity. However, the images are not entirely clear. It is very important to resolve whether patches devoid of trichomes are indeed present in the A6 tergite, because, if so, these patches would indicate that iab-6 is able to bypass the HS1 insulator to some extent. These same HS1 insertions into F7attP50 present a second problem for the reader: A5 and A6 are pigmented, indicating that iab-5 is also able to bypass the HS1 insulator. In the absence of clarification here, many readers will be mystified and discouraged from trying to make sense of what is a rather complicated paper.iab-6 and iab-7 during the initiation phase—and rescuing the GOF transformation of A6A7—is likely different than blocking iab-6 enhancers from activating Abd-B in PS11/A6. That this is likely the case in this instance is suggested by the sternite phenotype. While there are bristles on the A6 sternite (which shouldn’t be there) the morphology isn’t identical to A5. Instead of a quadrilateral shape, the sternite still has hints of the “banana” shape. This is also true when Fab-7 is replaced by a 209 bp fragment from Fab-8 that has blocking activity but lacks full bypass activity—which is conferred by a neighboring 165 bp fragment. In other replacements, such x5Pita the A6 sternite looks like A5. In the x5Pita replacement, the trichome pattern in A5 resembles A4 in being more densely packed and the lack of small patches of naked cuticle.In WT the trichome lawn in A5 is not always uniform and we often observe small patches of naked cuticle. So it wouldn’t be surprising to see small patches of naked cuticle in A6 in replacements that lack bypass activity. On the other hand, blocking crosstalk between Figure 5: Contrary to the text, in part C, ventral nerve cord expression of HS2505R-HS1248R-SV40 and HS2505R-SV40-HS1248R look very similar to one another and to HS2505R-HS1248R. In addition, Abd-B expression in the ventral nerve cord should be shown for the HS2505R-mSV40-HS1248R genotype.505R-HS1248R, HS2505R-HS1248R-SV40HS2 and 505R-SV40HS2-248RHS1 embryos. Figure 5—figure supplement 1 shows that the intensity of the signal is lower in PS11/A6 in 505R-SV40HS2-248RHS1 compared to both 505R-HS1248RHS2 and 505R-HS1248R-SV40.HS2We agree that difference in expression between these strains is hard to see with naked eye. We added supplementary Figure 5—figure supplement 1 that shows fluorescence intensity plot profiles of In part E, the right-hand genotype I believe should be HS2505R-HS1248R.Error is fixed.Page 20: Why reference Figure 2B here? Shouldn't this be 5E? The proper reference for the phenotypes of bxd and pbx mutations is definitely not Bender et al. – it should be one of the older Ed Lewis references!!!The reference is fixed, the proper citations have been added.Page 23: "activate Ubx expression in PS5/A1" should be "activate expression in PS6/A1". Also "is abrogated in PS6/A1 and more anterior parasegments/segments" should be "abrogated in PS6/A1 and more posterior parasegments/segments".The typos are fixed.Page 26 top: "In the Fub-1 deletion, the proximal endpoint of the bxd/pbx TAD shifts to the Ubx promoter region". I don't see this shift in Figure 2 or in the schematic in Figure 7. Explain what is meant here or delete.We refer to a subTAD formed by Fub-1 and bxd PRE. Clarification was added to the text.In addition, "There could be several reasons for this discrepancy" is quite unclear. What discrepancy? Is it that one might expect the WT and F1attP data to look the same? The writing badly needs clarification here.rd of the embryos. On the other hand, (a) not all of the cells in the posterior have detectable F1HS2 transcripts and (b) transcription is not continuous—but instead comes in bursts. Thus, additional explanation is that the disruption of boundary function is also not continuous, but rather linked to the bursts. We hope this point is now clear.The point we are trying to make is that a TAD-like MicroC pattern is observed in WT—even though boundary function should, in principle, be disrupted in ~2/3rds of the cells in the embryo. The TAD-like signal we observed could be from the remaining 1/3Page 27: "In this case, iab-6 would drive HS2 expression in PS11(A7) cells" should be "In this case, iab-6 would drive HS2 expression in PS11(A6) cells".Typo is fixed.Reviewer #2 (Recommendations for the authors):Overall, the work was well planned and conducted, and the results are clearly presented and interpreted. As mentioned in the public review, my main question is how general and significant the mechanism is in insulator function and animal development. Below are specific points.1. Active transcription and chromatin boundaries are often found in close proximity. Can the authors check their micro-C and available PolII ChIP data from wt embryos to globally assess other known/novel boundary elements that show similar organization: an insulator (based on the presence of CTCF or other insulator protein binding sites) is near an active promoter that can transcribe through the insulator. In this way, can they predict other loci that may use a similar strategy in insulator regulation?This is a good idea and we are planning to do this type of analysis. Probably the most interesting possibility is that these events will be tissue/cell type specific—which is what we see here. This will complicate the analysis in flies  unless one can isolate specific tissues/cell types and do both microC and RNA-seq.2. In Figures 4 and 5, the authors nicely showed that depleting HS2, adding the SV40 terminator, and changing the direction of the promoter all restore the insulator activity of HS1. Although these manipulations are supposed to remove or disrupt the transcriptional read-through of the non-coding transcript, the authors should validate the assumption. The authors can perform PolII ChIP-qPCR on some of the transgenes to directly show that the read-through is gone, for example in the HS2505R-SV40-HS1248R and the HS1248-HS2505R animals (and HS2505R- HS1248R) as a control.We are planning to conduct this study using transgenes.3. Many promoters  are divergent, which means producing transcripts in both directions. However, the directionality of the HS2 promoter seems very strong, and maintaining such directionality is important for the function of HS2. Can the authors comment on what may ensure the directionality of the HS2 promoter?We didn’t detect transcripts from HS2 in WT going the opposite direction. However, the reason could be that transcription in the “forward” direction occurs at a much higher frequency than in the “reverse” orientation. So if you are looking by deep sequencing you can detect the reverse transcripts, while they are no readily detected using in situ hybridization.4. There are a few typos in the text, and some labeling mistakes for the figures .All typos are fixed.


Reversal of fetal hemoglobin (HbF) silencing can ameliorate the effects of sickle cell anemia. Despite available gene therapy and stem cell transplantation modalities, the majority of affected patients worldwide will not have access to these in the near future. Thus, there is a need for safe and effective small-molecule therapeutics. We report here that stable occupancy of a major HbF silencing complex containing BCL11A, MBD2a–NURD, and PRMT5 and exclusion of the transcriptional activator NF-Y at the γ-globin gene promoter require specific features of MBD2a. These results provide a unified model for the relationships between the previously reported HbF silencers MBD2–NuRD, BCL11A, DNA methylation, and PRMT5 that may facilitate development of therapeutic agents to reverse HbF silencing. During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2–NuRD. In this report, we present direct evidence that MBD2–NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a–NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine–binding domain (MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a–NuRD, PRMT5, and DNA methylation in HbF silencing. The study of vertebrate globin gene switching during erythroid development has provided many primary insights into higher eukaryotic gene regulation. In humans, the switch in the β-globin gene locus involves sequential activation followed by silencing of the ε- and γ-globin genes HBG1 and HBG2) during embryonic and fetal erythroid stages, respectively, resulting in predominantly β-globin gene expression, and hence hemoglobin A (HbA), shortly after birth. A number of known transcription factors are critical for mediating silencing of the fetal γ-globin genes in adult human erythroid cells, including BCL11A, LRF/ZBTB7A, EKLF/KLF1, and ZNF410, among others  and HBG2. In addi6The role of MBD2 and its associated NuRD corepressor complex in embryonic/fetal β-type globin gene silencing has been established in our previous studies in both transgenic animal and human erythroid cell models. In the transgenic animal model in which an entire human β-globin locus was inserted, this effect appeared to be largely dependent on DNA methylation , 9, 10. In an effort to elucidate the basis for the observation that the MBD2–NuRD complex and not the MBD3–NuRD complex is required for full silencing of the γ-globin gene in adult human erythroid cells, studies of its occupancy at the promoter and its effects on nucleosome positioning and chromatin structure, as well as its interactions with other known silencing factors, were performed. Our findings indicate that MBD2 occupies the methylated HBG promoters in adult erythroid cells in which these gene are silenced and support the concept that MBD2 and BCL11A act as cofactors in the same silencing complex. These studies reveal that the MBD2a isoform is more capable than MBD2b in mediating γ-globin silencing due to a combination of the preference of its methyl cytosine–binding domain (MBD) for the methylated HBG promoters and its N-terminal arginine–rich GR domain, which together impart high-affinity binding to methylated promoter DNA. In addition, the GR domain recruits MEP50/PRMT5 to the promoter, which contributes to a closed chromatin conformation and silencing.SI Appendix, Table S1) in the HUDEP-2 erythroid cell line , in parental and MBD2KO cells, consistent with the same nucleosome positioning at both loci.ChIP-qPCR assays .MBD2 differs from MBD3 in that the former has a much higher affinity for DNA-containing methylated CpG dinucleotides A 19). M. M19). M+ progenitor–derived primary erythroid cells . In order to determine the potential roles of the MBD2-binding domain and the GR domain in HBG silencing, in vitro binding studies were carried out with methylated HBG promoter sequences containing two methylated CpG sites at positions −53 and −50 relative to the TSS. For these studies, isothermal calorimetry assays were performed with peptides containing the MBD2 MBD with or without the GR domain. Additional assays were carried out with a peptide containing the GR domain and an MBD2 MBD domain with a Y178H mutation, which decreases its binding preference for methylated cytosines . As expected, the MBD does not bind to unmethylated DNA, consistent with the 100-fold selectivity for methylated DNA that was reported previously  (D = 75 ± 8 nM). Hence, the GR domain increases the affinity by 10-fold. In contrast, the binding affinity of the GR domain plus wild-type MBD peptide for methylated promoter sequences containing the −162 methyl CpG (KD = 540 ± 200 nM) was much lower than for the −53/−50 CpG–containing sequences (As shown in ig. S5C) –23. By fequences C. NotablD = 77,000 ± 22,000 nM, SI Appendix, Fig. S5A). At the same concentrations that we used for wild-type protein, MBD2-MBD (Y178H) did not bind unmethylated DNA, but at higher concentrations, we could detect very weak binding , compatible with approximately fivefold selectivity for methylated DNA. This selectivity is much lower than for the unmutated domain and is comparable to the effects of the Y178F mutation studied previously (D = 164 ± 14 nM), indicating that the GR domain partially compensates for the Y178H mutation.To test whether methylation selectivity of the MBD contributes to the high-affinity association and gene silencing, we mutated a tyrosine residue (Y178) that directly interacts with a methyl-cytosine residue and plays a central role in preferential binding to methylated DNA –24. We ceviously , 23. To SI Appendix, Fig. S3A). To determine the importance of the methylated cytosine–binding preference of MBD2a for binding to the promoter and silencing, site-specific base editing of the endogenous MBD2 MBD was carried out in HUDEP-2 cells to introduce the Y178H mutation in the MBD2 MBD domain . As shown in N = 47) had elevated levels of the percentage of γ/(γ+β) RNA which is the result of an increased γ-globin RNA level , compared with wild-type control cell clones (N = 26). The effect was highly statistically significant but variable, with the percentage of γ/(γ+β) mRNA levels ranging from 0.13% to 34.24% compared to a range from 0.04% to 1.00% in control cell clones. The MBD2 protein level in wild-type clones and edited clones is equivalent to the level in ABE-sgSCR control HUDEP-2 cells . Notably, the chemical shifts do not follow a linear change based on DNA concentration. Instead, many affected peaks shift in different directions as the protein concentration exceeds that of the DNA. This observation suggests that when in excess, more than one GR domain weakly interacts with the same DNA.To further assess the structure and DNA-binding properties of the GR domain, we collected 2D-15N-HSQC spectra for the isolated MBD and GR-MBD proteins bound to the same methylated DNA. As shown in SI Appendix, Fig. S7C, the GR domain shows characteristics of an unstructured domain even in the presence of the MBD and when bound to DNA. The chemical shifts are essentially identical to that of the isolated domain. Likewise, the MBD shows similar chemical shifts when bound to DNA with or without the GR domain . These data indicate that the GR domain behaves as a fuzzy DNA–binding region, promoting DNA binding without adopting a regular structure. Our previous studies showed that the central intrinsically disordered region of MBD2 behaves similarly  followed by affinity purification. As shown in SI Appendix, Fig. S8).Further studies were carried out to investigate whether or not the GR domain recruits additional factors involved in γ-globin gene silencing in addition to its role in increasing the binding affinity of MBD2a for methylated HBG promoter sequences. Unbiased subtraction proteomic analysis was performed on nuclear proteins immunoprecipitated by enforced expression of TAP-tagged MBD2a or MBD2b in MBD2KO HUDEP-2 cells . As histone H3R8 symmetric dimethylation is considered a repressive mark for gene expression mediated by MEP50/PRMT5, we performed ChIP-qPCR assays of symmetrically dimethylated H3R8 histones (H3R8me2s), which showed high level of H3R8me2s occupancy at the HBG promoter in parental HUDEP-2 cells that was significantly diminished in MBD2 KO HUDEP-2 cells . PRMT5 knockdown (PRMT5KD) resulted in 20-fold and fourfold increases in γ-globin mRNA levels, respectively, in HUDEP-2 and CD34+ cells .It is notable that the nucleosome positioned at the promoter in parental HUDEP-2 cells, in which HbF expression is silent, is located in a position that would block access of both the key transcriptional activator complex NF-Y, whose preferred binding site is at nucleotide position −89 relative to the TSS, and the transcription initiation complex at the TSS, but would not block access for BCL11A to bind at its preferred binding site at nucleotide position −115. Moreover, the NF-YA DNA-binding domain is not of the type that has been shown to compete successfully with nucleosomes for DNA occupancy . ConsistMBD2 differs from MBD3 largely by the fact that its unique MBD imparts highly preferential binding to methylated CpG-rich DNA –21. SomaTwo approaches were taken to determine the contributions of the high-affinity methyl CpG–binding domain and the GR domain of MBD2a to HBG gene silencing. First, a series of detailed in vitro binding assays indicated that high-affinity binding of the MBD2-specific MBD to methylated HBG promoter sequences required the presence of the GR domain and a wild-type MBD since either absence of the GR domain or introduction of a Y178H amino acid substitution in the MBD resulted in decreased affinity. The functional importance of the wild-type MBD2 MBD for γ-globin gene silencing in adult erythroid cells was confirmed by editing the endogenous MBD2 gene in HUDEP-2 cells to introduce the same Y178H mutation that diminishes in vitro binding. This resulted in a highly statistically significant but variable increase in the percentage of γ/(γ+β) mRNA levels among individual edited clones, ranging from 0.1% to 34%. We interpret this variability to be reflective of the fact that the Y178H mutation only modestly reduces the binding affinity of MBD2-GRMBD region. This suggests that promoter methylation is required for the highest binding affinity, and thus highest probability of MBD2a–NuRD occupancy at the HBG promoter, which in turn is required for complete silencing. Since there is a relative paucity of methylated CpGs in the promoter compared to CpG islands, it seems likely that stable occupancy of the MBD2a–NuRD complex requires binding of BCL11A, as reflected by the same degree and pattern of chromatin opening and increased level of HBG transcription when either is depleted , 18. The+ progenitor–derived primary erythroid cells, but resulted in only a minimal further increase in MBD2KO HUDEP-2 cells. Thus, PRMT5 occupancy and the majority of its silencing effect are dependent on the presence of MBD2a–NuRD at the promoter. PRMT5 is known to symmetrically methylate arginine in histones H3 and H4 to facilitate inactive chromatin formation  as described previously , IL-3 was omitted from the culture medium. In the third phase, SCF was omitted from day 16 to day 21. The cell concentration was adjusted to 106 cell/mL on day 11 and to 5 × 106 cell/mL on day 15. The cells were cultured at 37 °C in the presence of 5% CO2.After isolation, human CD34escribed . Cells wSI Appendix, Methods.HUDEP-2 cells were maintained and expanded by the method described previously , 16 and SI Appendix, Methods.Lentivirus constructs were packaged into 293T cells and collected for transduction in HUDEP-2 or CD34+ progenitor primary cells as previously described . DetailepLV203 vectors containing sequences encoding MBD2sgR-TAPtag or MBD2b-TAPtag were packaged as described previously  and usedSI Appendix, Table S5, and were designed to generate PCR products of ~100 bp fragment. The quality of ChIP was verified by real-time PCR. Details of the method are provided in SI Appendix, Methods.Chromatin immunoprecipitation-qPCR assays were performed as described  with minSI Appendix, Methods.ATAC-seq was performed as previously described . A totalSI Appendix, Table S5.Nuclei from 250,000 HUDEP-2 cells were isolated as previously described . ThereafThe sequence of adenine base editors NG-ABE8e was cloned into lentiCRISPRv2 to generate a lenti-NG-ABE8e lentiviral vector. The oligos of gRNA sequence targeting the MBD2 Y178 codon were synthesized by IDT. After annealing into dsDNA, the DNA fragment was ligated with lenti-NG-ABE8e vector, followed by lentiviral preparation and transduction. Infected HUDEP-2 cells were selected by puromycin for 3 d post transduction. DNA was extracted for editing efficiency testing by PCR and Sanger sequencing. The selected stable transfected cells were seeded in a 96-well plate for single-colony growth. The single-colony-cloned cells were tested by the same PCR/sequencing method to determine editing efficacy. Both edited clones and unedited clones were differentiated in parallel for 3 d and mRNA harvested for qPCR to quantitate globin gene expression.SI Appendix, Methods.The gel bands from control and MBD2b-TAPtag and MBD2-TAPtag pulldowns containing all proteins from each sample were excised from the gel, cut into equal-sized cubes (approximately 1 mm), and transferred to a siliconized Eppendorf tube. The detailed method for sample preparation is provided in SI Appendix, Methods.Samples were analyzed using the LC–MS system which consisted of a Thermo Electron Q-Exactive HF-X mass spectrometer with an Easyspray Ion source connected to an Acclaim PepMap 75 µm × 2 cm nanoviper C18 3 µm × 100 Å precolumn in series with an Acclaim PepMap RSLC 75 µm × 50 cm C18 2 µm bead size (Thermo Scientific). The data were analyzed by database searching using the Sequest HT search algorithm using a custom human database downloaded from Swiss Pro. The following variable modifications were considered: oxidized methionine (+16 m/z) and carbamidomethyl cysteine (+57 m/z). Details of the LC–MS methodology and proteomics data analysis are provided in Appendix 01 (PDF)Click here for additional data file.Dataset S01 (XLSX)Click here for additional data file.


IRF1 in leukemia, we explored the potential of IRF1 expression level as a stratification marker for human acute myeloid leukemia. We show that IRF1-based stratification identifies distinct cancer-related signatures in patient subgroups. These findings establish IRF1 as a pivotal HSC controller and provide previously unknown insights into HSC regulation, with potential implications to IRF1 functions in the context of leukemia.Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between blood cell production and self-renewal. While inflammation-related signaling is a critical regulator of HSC activity, the underlying mechanisms and the precise functions of specific factors under steady-state and stress conditions remain incompletely understood. We investigated the role of interferon regulatory factor 1 (IRF1), a transcription factor that is affected by multiple inflammatory stimuli, in HSC regulation. Our findings demonstrate that the loss of IRF1 from mouse HSCs significantly impairs self-renewal, increases stress-induced proliferation, and confers resistance to apoptosis. In addition, given the frequent abnormal expression of  IRF1 controls essential murine HSC functions and provides a way to stratify human AML. Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic hierarchy and ensure life-long blood cell production through their mainly quiescent nature, multilineage differentiation potential, and self-renewal ability (Interferon (IFN) regulatory factor 1 (IRF1) is a transcription factor (TF) with a central role in innate and adaptive immune responses , type I and type II IFNs, interleukin-1 (IL-1), IL-12, and granulocyte colony-stimulating factor  cells, concomitant with increased numbers of CD4+ T cells (−/−Irf1 mice display increased frequencies of granulocytic precursors with impaired granulocytic development in the bone marrow (BM)  and myelodysplastic syndrome (MDS) with aberrations of chromosome 5, where the −/−Irf1 mice compared to wild-type (WT) controls (using the gating strategy outlined in fig. S1A-C). Consistent with previous studies  CD150+ CD48−] and LSK CD150+ CD48+ cells  revealed 169 up-regulated and 134 down-regulated genes in −/−Irf1 HSCs compared to WT HSCs (−/−Irf1 HSCs showed suppression of IFN-α and IFN-γ responses (−/−Irf1 HSCs exhibited diminished unfolded protein response (UPR) and down-regulation of components associated with key cellular integrity functions, such as the spliceosome, multiple DNA repair pathways, and epigenetic regulation (Irf1−/− HSCs displayed down-regulation of genes near p300 DNA binding sites (fig. S5C and table S6). Despite no significant differences in HSC cell cycle status in primary −/−Irf1 mice (−/−Irf1 HSCs (fig. S5D). This finding may be linked to the observed reduction in LPS-induced HSC proliferation in primary −/−Irf1 mice (We next performed bulk RNA sequencing (RNA-seq) of HSCs from WT and −/− HSCs . Differe−/− mice , the RNA−/− mice .−/−Irf1 HSCs /STAT3 signaling gene set  and cont−/− HSCs , suggestignaling . These r−/−Irf1 HSCs and the challenge in functionally determining HSC lineage priming in primary and transplanted mice due to the strong downstream influences, we also examined enrichment of cell type–specific gene sets in WT and −/−Irf1 HSCs. −/−Irf1 HSCs up-regulated both lymphoid (CLP) and myeloid (PreGM) genes . Nonetheless, enrichment of HSC gene sets, often derived from comparisons between HSCs and more differentiated progenitors, does not necessarily indicate improved function, as shown by observations between aged WT HSCs versus their younger counterparts (Stat1−/− HSCs (−/−Irf1 HSCs (fig. S5F). Collectively, these findings suggest a marked deviation in the transcriptional profile of −/−Irf1 HSCs consistent with a compromised HSC function.Considering the increased CD150 expression on nt cells . Moreove−/−Irf1 cells on HSCs cannot be excluded. Nevertheless, our findings provide valuable insights into IRF1-regulated genes in HSCs that were not subject to transplantation stress, revealing reduced functionality, blunted apoptosis, and altered immune signaling in −/−Irf1 HSCs.It is important to acknowledge that these transcriptional findings were obtained in the context of a whole-body IRF1 deficiency, where the influence of nonhematopoietic and hematopoietic −/−Irf1 HSCs in primary mice and could contribute to their maintenance at early time points after transplantation. To experimentally validate this transcriptional finding, we measured basal apoptosis in HSCs using annexin V staining and flow cytometry. Consistent with the transcriptomic analysis, −/−Irf1 HSCs displayed reduced annexin V staining compared to WT controls . MHC class II expression was recently linked to HSC functionality . The AML samples were allocated into three groups: IRF1high (the top 25% expressing samples), IRF1low (bottom 25% expressing samples), and IRF1medium for the remaining samples  (fig. S7A). In addition, IRF1low AML enriched for patients with t and t translocations and had fewer patients with a normal karyotype (NN). The IRF1high group, on the other hand, had fewer patients with chromosome inversion 16. Furthermore, IRF1low and IRF1high samples displayed differences in the French-American-British (FAB) classification of AML . These results imply that IRF1-based stratification can be used to identify AML subgroups with distinct underlying cellular mechanisms.Transcriptomic analysis revealed that IRF1IRF1 expression among various human leukemia subclasses (GSE13159) revealed significant down-regulation of IRF1 in most leukemia subclasses compared to healthy BM (fig. S7C). However, some lymphoid leukemias exhibited significant up-regulation of IRF1, and IRF1 expression varied substantially between patients within all leukemia subclasses. While further studies are required to decipher the role of IRF1 in leukemia and the relationship between IRF1 expression and clinical features, such as overall survival and therapy response, our results suggest the potential of IRF1-based patient stratification for identification of AML subgroups with distinct disease features.Last, comparison of IRF1 expression.IRF1 has been extensively studied in mature blood cells, particularly in the context of inflammation, and was shown to control various cellular processes, including apoptosis, proliferation, and expression of MHC class I and II molecules. However, its role in the HSC compartment was barely explored. Nevertheless, several inflammatory mediators that signal via IRF1 affect HSC activity . Reverse transplantations uncovered that the Irf1−/− environment also repressed CD8+ T, NK, and B lymphoid cell production. The transplantation of −/−Irf1 cells into WT recipients, but not −/−Irf1 recipients, triggered an expansion of −/−Irf1 B cells. This expansion may be linked to previous observations of intrinsically regulated −/−Irf1 B cell expansion in response to infectious stress  mice to examine the role of IRF1 in HSCs and acknowledge the potential influence of nonhematopoietic cells in the observed phenotypes. Utilization of conditional KO models for IRF1 will be valuable for further investigations, enabling exploration of potential interactions between hematopoietic and nonhematopoietic cells in driving the functional changes in IRF1 is frequently observed in MDS and AML and increases tumor predisposition and mutation rates in mice when combined with other cancer-promoting mutations  being investigated. Nevertheless, our findings suggest that IRF1 expression can be used to identify subclasses of leukemia with diverse underlying mechanisms and clinical characteristics, including cancer-associated gene signatures, differences in cell proliferation, survival, DNA damage responses, and differentiation, as well as in karyotype and cytogenetic risk distribution. Moreover, our observation that IRF1 expression differs substantially between patients within various leukemia subclasses supports the use of IRF1 as a broad leukemia stratification marker.Down-regulation of Irf1−/− (B6.129S2-Irf1tm1Mak/J), C57Bl/6, and B6.SJL-PtprxaPepcb/Boy mice were used throughout the study. The mice were maintained at the University of California San Diego Leichtag building vivarium. All procedures were conducted in accordance with ethical permit S00218 approved by the Institutional Animal Care and Use Committee. Three-month-old male mice were used for the primary phenotyping analyses. Four-month-old male mice were used for steady-state cell cycle analysis. Three-month-old female mice were used for LPS-induced cell cycle examination of primary mice. Eight-week-old male donors and 14-week-old female recipients were used for cell cycle analysis in chimeras. Two-month-old female donors and recipients were used for competitive HSC transplantation experiments. Three-month-old female recipients, 6-week-old WT and Irf1−/− CD45.2+ male donors, and 3-month-old CD45.1+ male competitive donors were used for reverse transplantations. Eight-week-old female mice were used for RNA-seq analysis. Five-month-old female mice were used for the apoptosis analysis. Two-month-old female mice were used for the ubiquitin assay. Three-month-old male mice were used for MHC class II expression analysis.Age- and gender-matched PB and BM were isolated and analyzed as previously described (Mice were administered 35 μg of LPS (Sigma Aldrich) intraperitoneally and euthanized 16 hours later for downstream analysis. Control mice were administered volume-equivalent 0.9% saline solution.−Sca-1+Kit+CD150+CD48− HSCs together with 300,000 whole BM (wBM) competitor cells. wBM chimeras were generated with 10 million total donor BM cells. Recipient mice were lethally irradiated with 9 gray and treated prophylactically with sulfamethoxazole and trimethoprim antibiotics for 2 weeks after transplantation in the drinking water. For serial transplantations, 276 donor HSCs were reisolated from each donor and transplanted together with 300,000 wBM competitor cells.Competitive HSC transplantations were performed using 100 LinLineage-depleted BM cells were stained with HSC-identifying antibodies before fixation and permeabilization with BD Perm/Wash buffer  at 4°C for 20 min. Cells were subsequently incubated with anti-Ki67 or immunoglobulin G 1 (IgG1) isotype control before being washed and resuspended in 7-aminoactinomycin D (10 μg/ml) diluted in Perm/Wash solution and incubated at 4°C overnight before analysis.BD Annexin V: FITC Detection Kit I (BD Pharmingen) was used according to the manufacturer’s instructions. Briefly, lineage-depleted BM cells were stained with HSC-defining antibodies before being washed and incubated with camptothecin at a final concentration of 5 μM for 5 hours at 37°C. The samples were then washed with ice-cold FACS buffer and resuspended in 1× annexin binding buffer to which annexin V and propidium iodide were added. The samples were vortexed and incubated in the dark for 15 min at room temperature. Additional annexin binding buffer was then added after which the samples were analyzed within 1 hour.After lineage depletion, antibody staining, and fixation, cells were incubated with anti–multi-ubiquitin antibody for 30 min at 4°C. The cells were then washed and resuspended with goat anti-mouse IgG Alexa Fluor 488 (Invitrogen) before analysis.Irf1−/− mouse into RLT lysis buffer supplemented with (1:100) β-mercaptoethanol. RNA was isolated using the Single Cell RNA Purification Kit (Norgen Biotek Corp.). cDNA generation and amplification were performed using the SMART-Seq v4 Ultra Low Input RNA Kit for sequencing (Takara Bio USA Inc.). Quality check was performed using High Sensitivity D5000 ScreenTape with TapeStation Analysis Software 3.2 (Agilent Technologies Inc.). Sequencing was performed using Nova Seq S4 . Preprocessing and analysis were done using The Galaxy platform . Cell type enrichment of up- and down-regulated genes was performed with CellRadar  with the lowest IRF1 expression were allocated to the IRF1low group. The middle expressing samples (n = 268) were defined as IRF1med. In addition, available data were used to assess karyotype, FAB score, and cytogenetic risk distribution between the groups. Gene expression profiles were used to subject the groups to global GSEA. Publicly available expression data from GSE13159 were used to evaluate the expression of IRF1 in various leukemia subclasses.Publicly available expression data from GSE6891 containing 537 human AML patient samples (<60 years of age) was used for patient stratification (t tests using Prism 9 (GraphPad software). Statistical details can be found in the figure legends. Division of mice into groups was randomized but not blind, and no statistical methods were used to determine the number of mice for the experiments. Multilineage (T + B + My) donor reconstitution levels below 1% in primary recipients were considered unsuccessful and excluded . Analysis of gene expression data is described above. Detailed information about antibodies, reagents, and instruments can be found in table S9.Statistical significance between experimental groups analyzed by flow cytometry was determined by unpaired Student’s