Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8. The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation.Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how  Lineage-determining transcription factors (TF) are master regulators of gene programs that frequently initiate self-reinforcing loops by autoactivation. TF autoactivation is important for cells to pass restriction points during development (referred to as points of no return) and to enforce cellular identity. Molecular circuitries of autoactivation have been studied for several TF, such as GATA-binding factor 1 (GATA1), PU.1 (encoded by Spi1), CCAAT enhancer-binding protein α and ε  and plasmacytoid DC (pDC)  development . IRF8 isDC (pDC) . Irf8 istivation , yet howIrf8 expression in hematopoietic cells is induced and maintained by enhancers located at –50 kb,+32 kb,+41 kb and +56 kb relative to Irf8 transcription start site (TSS)  . Enhanceour work . Enhanceour work . EnhanceIrf8 regulates DC and monocyte specification in a dose-dependent manner  in the following referred to as lncIrf8 and a cDC1 specific lncRNA  Capture-C in MPP, CDP, pDC, cDC1, and cDC2  is important for regulation of chromatin conformation through loop extrusion  and we tus in DC . Interesnhancers  are locarf8 gene . There acription .lncIrf8 promoter is high, but this locus shows less interactions with the Irf8 promoter in pDC compared to CDP, cDC1 and cDC2 , which is in line with the general characteristics of lncRNA. Then second, we deleted 300 bp in the lncIrf8 promoter by CRISPR/Cas9 editing in conditionally immortalized HoxB8 MPP  overexpressed lncIrf8 in wild-type MPP and (ii) re-expressed lncIrf8 in lncIrf8 promoter KO MPP and monitored its impact on DC development  also compromised pDC and cDC1 development  transcribed from the Irf8 +32 kb enhancer. lncIrf8 itself lacks activities in DC differentiation but a lncIrf8 promoter element is crucial for pDC and cDC1 development. Upon deletion of this lncIrf8 promoter element pDC and cDC1 development was severely compromised, demonstrating that this sequence is important for both pDC and cDC1 development.elopment . Frequenelopment , and herIrf8 expression during DC differentiation is in a first step initiated and activated through flanking enhancers, including the +32 kb enhancer by autoactivation  with partner TF that bind to specific DNA sequences  were obtained from mouse bone marrow and expanded in vitro with a two-step protocol as described in HoxB8 MPP were differentiated into DC using a two-step protocol modified from - CD117hi CD135low/-, CDP are Gr1- CD117int CD135+ CD115+, cDC1 are CD11c+ CD11blow/- XCR1+, cDC2 are CD11c+ CD11b+ XCR1-, pDC are CD11c+ CD11b- B220+.Wild-type bone marrow cell-derived MPP, CDP, cDC1, cDC2, and pDC were generated in vitro with the two-step protocol as described in The chromatin conformation capture (3 C) library preparation protocol used in this study was modified from Irf8 promoter viewpoint 2 oligonucleotide probes targeting Irf8 promoter were designed with the design tool Oligo (https://oligo.readthedocs.io/en/latest/index.html). Oligonucleotide probes are positioned adjacent to the DpnII cut sites on a restriction fragment spanning the Irf8 promoter  and 70 bp ssDNA biotinylated oligonucleotides were synthetized by Sigma-Aldrich (listed in Appendix 1-key resources table).For Irf8 promoter viewpoint, NuTi Capture-C was performed as previously described  pipeline . For visualization of lncRNA expression, we created a custom reference genome of mm9 by following the tutorial: available here. Cellranger pipeline was then processed to generate an expression count matrix of pDC. We next used scanpy  and pMD2.G (Addgene #12259) for lentiviral packaging and envelope expressing plasmids were kind gifts from Didier Trono, EPFL, Lausanne, Switzerland. The gRNA expressing vector pLKO5.sgRNA.EFS.tRFP (Addgene #57823) and pLKO5.sgRNA.EFS.tRFP657 (Addgene #57824) were kind gifts from Benjamin Ebert, Harvard Medical School, Boston, USA .lncIrf8 overexpression and rescue, lncIrf8 cDNA was introduced into polyA containing lentivirus vector pGFP-pA to generate plncIrf8-pA. pGFP-pA was constructed from pCIG3  (For  #61422)  and pTet #61422) , respectlncIrf8, dCas9-VP64-GFP, and KRAB-dCas9-GFP were delivered into HoxB8 MPP by lentiviral infection. Briefly, lentiviral particles were produced by calcium phosphate transfection  HoxB8 MPP by CRISPR/Cas9. Briefly, pairs of gRNAs each for lncIrf8 promoter KO and cDC1 specific +32 kb enhancer KO (Appendix 1-key resources table) were designed with the IDT online gRNA design tool. For cDC1 specific +32 kb enhancer KO we additionally used the same gRNA sequences from Mx-Cas9-GFP HoxB8 MPP were used to generate One 10 cm dish (Bio-One) with 1.8 million HEK293T cells (70%–80% confluency) in 10 ml DMEM plus supplements (see above) was used to produce gRNA expressing lentivirus particles. HEK293T cells were transfected with 10 μg gRNA vector, 7.5 μg psPAX2, and 2.5 μg pMD2.G per gRNA by calcium phosphate transfection and lentivirus particles were harvested 48 and 72 h after transfection.+ RFP+ RFP657+ cells). Single-cell clones were obtained by single-cell FACS sorting or limiting dilution.The gRNA expressing lentiviral particles were used to infect 3 million Mx-Cas9-GFP HoxB8 MPP. Cas9 and GFP expression were induced by mIFNα  for 4 h, followed by FACS sorting for cells expressing the two gRNAs and Cas9 (GFPhttps://eu.idtdna.com/site/order/designtool/in-dex/CRISPR _SEQUENCE). For lncIrf8 promoter KO, 5 out of 71 single-cell colonies with homozygous deletions were subjected to off-target analysis. The top 2–5 predicted coding or non-coding targets were selected and HoxB8 MPP clones without off-target effects, or where off-target effects occurred in genes that were not expressed in MPP, CDP and DC subsets, were used for further studies , each consisting of 0.75 million HEK293T cells (70–80% confluency) in 5 ml DMEM with supplements (see above). HEK293T cells were transfected with 5 μg plncIrf8-pA or pGFP-pA, 2.5 μg psPAX2, and 2.5 μg pMD2.G. Lentivirus particles were concentrated as above and used to infect 0.5 million HoxB8 MPP; single-cell clones were generated by limiting dilution and genotyped using the primers listed in Appendix 1-key resources table. Two out of 47 HoxB8 MPP colonies with plncIrf8-pA and 3 out of 19 HoxB8 MPP colonies with pGFP-pA (control) were expanded and subjected to Flt3L-directed DC differentiation.lncIrf8 knockout rescue was carried out in lncIrf8 promoter KO HoxB8 MPP. FACS sorted cells that genotyped as lncIrf8 promoter KO homozygous deletion cells, were infected with lentiviral particles expressing lncIrf8. Lentiviral infection conditions were the same as for lncIrf8 overexpression in wild-type HoxB8 MPP (see above).lncIrf8 overexpression experiments. In brief, virus particles from ten 6 cm dishes were used to infect 0.5 million wild-type HoxB8 MPP, followed by FACS sorting for GFP+ cells expressing dCas9-VP64 or dCas9-KRAB. Doxycycline (1 μg/ml) was used to induce dCas9-KRAB expression 2 days before cell sorting.CRISPR activation and CRISPR interference were performed by infecting wild-type HoxB8 MPP with lentiviral particles expressing dCAS9-VP64_GFP and pTet-KRAB-dCas9-GFP, respectively. The virus particles were produced as in the lncIrf8 and Irf8 promoters were cloned into pLKO5.sgRNA.EFS.tRFP as above. The dCas9-VP64-GFP and dCas9-KRAB-GFP HoxB8 MPP were then infected with specific gRNAs for gene activation and repression. The conditions for gRNA infection were the same as in lncIrf8 promoter KO experiments. Doxycycline (1 μg/ml) was given to the sorted dCas9-KRAB-GFP cells every 3 days to ensure sustained dCas9-KRAB expression.gRNAs targeting https://software.broadinstitute.org/morpheus/).DC subsets were analyzed by flow cytometry using FACS Canto II or LSR Fortessa (both from BD Biosciences). The information for flow cytometry antibodies is shown in Appendix 1-key resources table. For live/dead staining, cells were incubated with 3 μl 7-AAD per test for 5–10 min before flow cytometry measurement. Cells were sorted by FACS Aria IIu or FACS Melody, and flow cytometry data were analyzed by FlowJo V10 . Data on DC frequencies were subjected to the hierarchical clustering and represented in heatmap format using the online tool MORPHEUS (lncIrf8 promoter KO and control HoxB8 MPP (single-cell clones) were expanded in vitro as described above. Cells were injected into recipient mice via the tail vein (5 million cells in 100 μl PBS per mouse). Donor cells from bone marrow and spleen were subjected to flow cytometry analysis at 7 and 14 days after cell transplantation.CD45.1 recipient mice were sublethal irradiated  1 day before transplantation. GAPDH was used for normalization of lncIrf8 and Irf8 gene expression.RNA was isolated by using the NucleoSpin RNA kit  according to the manufacturer’s instructions. RNA was subjected to cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit  with Murine RNase Inhibitor  (Appendix 1-key resources table). RT-qPCR was performed by a StepOnePlus Real-Time PCR system (Applied Biosystems) with SYBR-green fluorescence . The primers for qPCR were listed in Appendix 1-key resources table. Mouse Tcons_00190250 (referred to as lncIrf8) and the cDC1-specific lncRNA Tcons_00190258. In brief, paired end 2×100 bps reads from RNA-seq of MPP, CDP, cDC1, cDC2, and pDC were aligned to mm9 genome using Star aligner (version 2.4) (lncIrf8 (Tcons_00190250) and Tcons_00190258 show low scores in both analyses, which faithfully supports their role as non-coding genes and exhibit low cross-species conservation.De-novo transcript assembly of RNA-seq data was used to search for unknown transcripts with no coding potential and this identified the pDC specific lncRNA ion 2.4)  and run ion 2.4) . Data weion 2.4) . The codion 2.4)  and Phylion 2.4) , respectlncIrf8, 3' and 5' end Rapid Amplification of cDNA Ends (RACE) PCR was performed using template-switching RT enzyme mix  and TA cloning kit  (Appendix 1-key resources table). The primers (listed in Appendix 1-key resources table) used for RACE PCR were synthesized by Eurofins Genomics except for 5' RACE-TSO, which was from IDT.To further characterize the major transcripts of Reverse transcript (RT) and template-switching: 4 μl (10 ng to 1 μg) total RNA (from DC differentiation day 5) were incubated at 80°C for 3 min and cooled rapidly on ice. RNA was then incubated with template-switching RT buffer, 1 mM dNTP, 5 mM DTT, 10 μM QT primer  and 1 μllncIrf8-F1 (10 μM) using Q5 high fidelity DNA polymerase. Second-round PCR: the PCR products from the first round PCR (1:20 dilution) was then used as template for the second round of PCR by using Q1 primer (10 μM), 3' RACE-GSP-lncIrf8-F2 (10 μM). Q0 and Q1 primers and the PCR programs are described in , 740609.50).First-round PCR: 1 μl of diluted sample was subjected to the first round PCR with Q0 primer and 3' RACE-GSP-2, 0.4 mM dATP and 1 μl Taq DNA polymerase in 25 μl was prepared and incubated at 70°C for 20 min for A-tailing of PCR products. Five μl of the A-tailed products were subjected to TA cloning into pCR2.1 vector according to the manufacturer’s instruction followed by Sanger sequencing.A-tailing and TA cloning: a reaction containing 5 μl of PCR clean-up product in Taq buffer, 1 mM MgCllncIrf8, template RNA from bone marrow cell-derived pDC was used to perform 5' RACE PCR using template-switching RT enzyme mix  according to the manufacturer’s instructions. Template switching was by 5' RACE-GSP-lncIrf8-R2 primer (10 μM) and template switching oligo (TSO) (75 μM). Similar to the 3' RACE PCR, two rounds of PCR were used to improve PCR specificity. In brief, 5' RACE-GSP-lncIrf8-R2 (10 μM) and TSO-specific primer (10 μM) were used to perform the first-round PCR, 5' RACE-GSP-lncIrf8-R1 primer (10 μM) and the TSO-specific primer (10 μM) were used to perform the second round PCR, followed by fragments A-tailing, TA cloning, and Sanger sequencing as described above for 3' RACE PCR.In order to identify the TSS of Irf8 +32 kb enhancer were predicted using a motif matching tool based on the MOODS (Transcription factor binding sites (TFBS) in the he MOODS  and posihe MOODS . The bitStatistical analyses were performed using Prism (GraphPad). Unpaired t test and Multiple t-tests were used to compare data from two groups, two-way ANOVA with Tukey’s multiple comparisons test was used to analyze data from more than two groups. Authors provide valuable evidence identifying a lncRNA transcribed specifically in the pDC subtype from the +32Kb promoter region which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. With convincing methodology, they provide in-depth analysis about the possible role of lncIrf8, and its promoter region and cross-talk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region that is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. The work will be of interest to immunologists working on immune cell development. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) 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:IRF8 enhancer element in negative feedback control of dendritic cell differentiation" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Satyajit Rath as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Stephen L Nutt (Reviewer #2).Thank you for submitting your article "A lncRNA identifies 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) Demonstrate the presence of lncRNA selectively in ex-vivo splenic pDC sub-type and confirm its expression in human pDCs.2) To clarify the role of irf8 in pdc development in regards to published data comparing the lncIrf8 and +32kb activity in their system: The authors conclude that lncIRF8 promoter KO hematopoietic progenitors are not capable of differentiating to pDCs . However, it is now understood that Irf8 is not essential for pDC development (see Sichien Immunity 2016 where they study WT:IRF8-/- BM chimeras) and that Irf8 deregulates some key markers, including CD11b. The authors need to account for this discrepancy. Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO, and if not, why does this phenotype differ from the Irf8 KO? In addition, it is essential that authors delete the +32 Kb enhancer region (as defined in Durai 2019) in their culture system and analyze the same with respect to +32Kb lncIRF8 promoter deletion in terms of expression of IRF8, lncIRF8 and its effect on the development of pDC vs cDC1.3) Authors should acknowledge/discuss clearly that one limitation of their study is that lncIRF8 might have other roles in DC/pDC biology which is yet unidentified in the current study.Reviewer #1 (Recommendations for the authors):1. While refining the cross-talk between promoter and enhancers authors have completely overlooked the functional significance of lncIRF8 in pDCs. Authors have demonstrated the expression of lncIRF8 is specific to pDCs, it is not evident whether all pDCs express it or a fraction of the same.a. Further authors should confirm the expression of lncRNA in ex vivo pDCs from various murine tissues. Is the expression of lncRNA also conserved in human pDCs?b. How is the expression of lncIRF8 modulated upon CpG stimulation/viral infection of pDCs?c. Is there any significance of lncIRF8 in pDC functions  these aspects could be studied without much effort as authors already have systems that can knockout lncIRF8 promoter or overexpress lncIRF8.2. Though authors have identified that lncIRF8 is specifically expressed in pDC subtype but while understanding its significance and regulation, it was preferred to study bulk DC cultures overlooking the finding regarding specificity. Experiments performed by employing dCas9 VP64/KRAB-GFP constructs, do provide some direction in terms of mechanistic understanding of the cross-talk between the promoter of IRF8 and lncIRF8. This data needs to be further strengthened by sorting pDC, cDC1 and cDC2 populations and studying the effect of the genetic manipulations on IRF8 and lncRNA expression in each cell type.3. One of the most important findings of the submitted study is to demonstrate that knocking out the lncIRF8 promoter also diminishes the expression of IRF8, pDC as well as cDC1 development . Once the IRF8 expression is suppressed the analysis of pDC and cDC1 differentiation is redundant. It is previously reported that the deletion of +32 Kb enhancer  in a mouse model did not affect pDC but specifically depleted cDC1 development. Authors should examine the deletion of only the +32 kb enhancer region in their culture system to sort out the hierarchy between the +32 Kb enhancer and +32 Kb lncRNA promoter in terms of DC subtype development (cDC1 Vs pDC). Does the deletion of the lncIRF8 promoter lead to epigenetic changes at the overall +32 region? It is expected that due to interactions and chromatin loop formation between IRF8 promoter with various enhancers elements; deletion of lncIRF8 promoter might play an important role in defining these interactions in a cell type-specific manner. These interactions become more valuable by the fact that activation of the lncIRF8 promoter leads to enhanced cDC1 differentiation. Does this simply mean more open chromatin conformation around the cDC1-specific +32 Kb region rather than correlating with the lncIRF8?Can authors analyze the epigenetic changes by performing similar experiments in some of the available pDC and cDC1 cell lines? As these cell lines are likely to have active IRF8 transcription and it would help to measure the effect of lncIRF8 promoter deletion/mutation/activation/repression on IRF8 expression and specific DC subtype maintenance.4. Based on the various earlier published reports and results from the current study authors have shown a schematic representation of the pDC and cDC1-specific interactions. Can these interactions be validated in primary/ex-vivo DCs and be correlated with lncIRF8 as well as IRF8 expression? Will it be possible for authors to validate these interactions in light of the lncIRF8 promoter deletion/mutation/activation/repression? Authors may be careful to perform these experiments in purified pDC and cDC1 fractions to get meaningful data.In case it is not possible to perform experiments with primary DCs/genetically modified HOXB8 cultures; if required, some of these experiments may be performed in the cell lines wherein overall interaction between various enhancer elements and promoters can be validated.5. Authors have also identified a TCONS_00190258 which is specifically expressed in the cDC1 subtype. Why not look at the expression levels of this transcript in the various experiments in the current study as enhancer element crosstalk will help in cell type-specific transcription of IRF8, lncIRF8, and TCONS_00190258?Reviewer #2 (Recommendations for the authors):I find the quality and interpretation of the genomics data to be excellent and I have no major concerns with that data. I do have some specific comments around the interpretation of the cellular data and the model presented.Specific comments1. The authors conclude that lncIRF8 promoter KO hematopoietic progenitors are not capable of differentiating to pDCs . However, it is now understood that Irf8 is not essential for pDC development (see Sichien Immunity 2016 where they study WT:IRF8-/- BM chimeras) and that Irf8 deregulates some key markers, including CD11b. The authors need to account for this discrepancy. Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO, and if not, why does this phenotype differ from the Irf8 KO?2. There does not appear to be compelling evidence that IRF8 binds in the lncIRF8 promoter in cDC1. Instead, IRF8 binds in the more 5' part of the +32kb enhancer identified by Durai 2019. How then is a putative IRF8 repressor complex linked to the lncIRF8 promoter as the authors propose? Moreover, the model does not appear to address why the IRF8 repressor complex binding to the IRF8 repressor complex would be active in cDC1 and not pDC.3. The authors use 2 versions of the HoxB8 MPP differentiation system (lines 207-234). Firstly, it is unclear, why two similar approaches are used, but more importantly, the second system outlined in figure 2 Suppl 3 shows that the lncIRF8 promoter is also required for cDC2 differentiation. This is a concern, as IRF8 is not expressed in cDC2, and Irf8-/- mice produce cDC2. Essential revisions:1) Demonstrate the presence of lncRNA selectively in ex-vivo splenic pDC sub-type and confirm its expression in human pDCs.Point 1: Presence of lncRNA selectively in ex-vivo splenic pDC sub-type.ex-vivo spleen pDC and bone marrow pDC were obtained from GSE114313 . The bulk RNA-seq dataset of splenic DC subsets pDC, cDC1 and cDC2 are from GSE188992 .This is interesting and we addressed this point by re-analyzing public scRNA-seq and bulk RNA-seq datasets for spleen. We also included the analysis of lncRNA expression in bone marrow. The scRNA-seq data of murine These new results are now shown in new Figure 1—figure supplement 3 (page 32) and described in the Results section, page 6, lines 151-154 as follows:“lncIrf8 and TCONS_00190258 show the same expression pattern in pDC and cDC1, respectively, in BM and spleen , as revealed by reanalyzing scRNA-seq and bulk RNA-seq data .”The Materials and methods section was updated accordingly .Nat. Immunol., 2018) were downloaded, BAM files were converted back into FASTQ files and a custom reference genome of mm9 was created now including the novel lncIrf8 (GenBank: ON134061) and Tcons_00190258 lncRNA (GenBank: ON134062). Data were subjected to bioinformatic processing as described in the Materials and methods section on page 17, lines 536-548 and visualized in UMAPs.More specifically, the scRNA-seq data from GSE114313  and spleen pDC . Expression of the cDC1 specific Tcons_00190258 lncRNA in BM and spleen pDC is very low to absent.In Dress et al., Front. Immunol., 2022). lncIrf8 and Tcons_00190258 lncRNA were visualized by IGV browser. As expected, lncIrf8 and Tcons_00190258 lncRNA were expressed in pDC and cDC1 of spleen, respectively, and the expression pattern was the same as in our in vitro bone marrow DC .To further extend this analysis, we analyzed the bulk RNA-seq dataset of splenic DC subsets pDC, cDC1 and cDC2 from GSE188992  signals .We found that the sequences of mouse onserved . The hum signals . The humle blood . Data oflncIrf8 is not conserved across species, which is in line with the general characteristics of lncRNA. We have now included this information in the Results section, page 7, lines 200-202.Taken together, the pDC specific 2) To clarify the role of irf8 in pdc development in regards to published data comparing the lncIrf8 and +32kb activity in their system: The authors conclude that lncIRF8 promoter KO hematopoietic progenitors are not capable of differentiating to pDCs . However, it is now understood that Irf8 is not essential for pDC development (see Sichien Immunity 2016 where they study WT:IRF8-/- BM chimeras) and that Irf8 deregulates some key markers, including CD11b. The authors need to account for this discrepancy. Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO, and if not, why does this phenotype differ from the Irf8 KO? In addition, it is essential that authors delete the +32 Kb enhancer region (as defined in Durai 2019) in their culture system and analyze the same with respect to +32Kb lncIRF8 promoter deletion in terms of expression of IRF8, lncIRF8 and its effect on the development of pDC vs cDC1.Point 1: Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO?Irf8-/- BM chimeras and the conclusions reached from their work that IRF8 is dispensable for pDC development. Previous studies, including the study of Sichien et al., 2016, showed that pDC are reduced in Irf8 knockout mice . Thus, there is the possibility that the conclusions reached by Sichien et al., 2016 on the impact of IRF8 on pDC function, is on the pDC population, which is left in the Irf8-/- mice. In addition the results in Sichien et al., 2016 are based on WT:Irf8-/- BM chimeras upon transplantation in lethally irradiated mice and thus the divergent results might be due to the experimental set-up WT:Irf8-/- BM chimeras versus Irf8 knockout mice.We appreciate the point raised and know the work by Sichien et al., 2016 on WT:lncIrf8 promoter KO we used immunophenotyping of CD11c+ CD11b- B220+ cells for in vitro pDC  and CD45.2+ Gr1- Siglec H+ cells for pDC following transplantation . Thus, we are confident to correctly detect pDC.Sichien et al., 2016, report on altered marker expression , which for CD11b and CD11c was not very prominent. In our study on the lncIrf8 promoter KO pDC cannot be measured because cells are absent in lncIrf8 promoter KO. Nevertheless, we visualized CD11c and CD11b expression on cells which are present by gating on single cells on day 7 and 9 of Flt3L directed DC differentiation in lncIrf8 promoter KO and control cultures. In lncIrf8 promoter KO cells CD11c expression was slightly reduced and CD11b expression was slightly increased . In parallel, we also designed a new pair of gRNAs to delete this +32 kb enhancer region. We followed the same procedure as in the lncIrf8 promoter KO experiments  for both the Durai et al. gRNAs and our new self-designed gRNAs to obtain homozygous clones of cDC1 specific +32 kb enhancer KO. Five out of 165 single-cell clones were subjected to DC differentiation and further analyzed at day 7 and 9. Frequencies of pDC were unaffected by +32 kb enhancer KO at the end of culture (day 9). There was some effect at day 7, however this was not statistically significant. The cDC1 specific +32 kb enhancer KO compromised cDC1 development at day 7 and day 9, which is in line with the phenotypes described by Durai et al., Nat. Immunol., 2019 and Murakami et al., Nat. Immunol., 2021 in mice.We followed the request of the reviewers and deleted the +32 kb enhancer region by using the same gRNAs as described in Durai et al., These new results are now shown in new Figure 2—figure supplement 4 in the revised manuscript . The Materials and methods section was also updated accordingly . Source data of new Figure 2—figure supplement 4B are also provided and described on page 36 lncIrf8 promoter KO affects both pDC and cDC1 development . This indicates that the lncIrf8 promoter element has further functions compared to the cDC1 specific +32 kb enhancer, e.g. to control pDC development. We have now indicated this point in the Discussion .In summary, our data show that deletion of cDC1 specific +32 kb enhancer affects cDC1 development while the 3) Provide evidence that IRF8 binds in the lncIRF8 promoter. Authors showed show DNA binding motifs for Irf8 in the lncIrf8, but no evidence of actual binding. Perhaps the +32kb enhancer is also physically involved, but either way more evidence is needed to fully support the authors' models. Instead, IRF8 binds in the more 5' part of the +32kb enhancer identified by Durai 2019. How then is a putative IRF8 repressor complex linked to the lncIRF8 promoter as the authors propose? Moreover, the model does not appear to address why the IRF8 repressor complex binding to the lncIRF8 would be active in cDC1 and not pDC.Irf8 during DC differentiation. The full +32 kb enhancer is delineated by the H3K27ac and H3K4me3 marks  and comprises both the +32 kb enhancer element by Durai et al. 2019 and the lncIrf8 promoter described here. Both elements appear to be important for full Irf8 +32 kb enhancer function in DC differentiation: (i) Deletion of the lncIrf8 promoter compromised both pDC and cDC1 differentiation ; (ii) Deletion of the +32 kb enhancer element by Durai et al. 2019 compromised cDC1 and to some extent also pDC differentiation ; (iii) positioning the dCas9-VP64 at the lncIrf8 promoter for CRISPR activation prominently enhanced cDC1 differentiation  and (iv) positioning the dCas9-KRAB at the lncIrf8 promoter for CRISPR interference compromised both pDC and cDC1 differentiation .We appreciate the reviewers’ point and can very well agree with the reviewers that the cDC1 specific +32 kb enhancer element by Durai et al. 2019 might also be involved in the negative feedback regulation of In our model we proposed that binding of the putative IRF8 repressor to the +32 kb enhancer is mainly in the 5’ part of the +32 kb enhancer, as demonstrated by the ChIP-seq of IRF8. There is a strong IRF8 binding signal detected in cDC and weak to no IRF8 binding signal in pDC .Thus, we propose the putative IRF8 repressor complex is only active in cDC and not pDC . We agree with the reviewers that at this stage of analysis we do not know why IRF8 binds to the 5’ part of the +32 kb enhancer in cDC but not in pDC. Addressing this point should be subject for further studies.4) Authors should acknowledge/discuss clearly that one limitation of their study is that lncIRF8 might have other roles in DC/pDC biology which is yet unidentified in the current study.lncIrf8 might have other roles in DC biology which is yet unidentified in the current study .We appreciate the suggestion made by the reviewer. We have now indicated that Reviewer #1 (Recommendations for the authors):1. While refining the cross-talk between promoter and enhancers authors have completely overlooked the functional significance of lncIRF8 in pDCs. Authors have demonstrated the expression of lncIRF8 is specific to pDCs, it is not evident whether all pDCs express it or a fraction of the same.a. Further authors should confirm the expression of lncRNA in ex vivo pDCs from various murine tissues. Is the expression of lncRNA also conserved in human pDCs?lncIrf8 and Tcons_00190258 lncRNA in ex vivo pDC is well taken. We addressed this point be re-analyzing scRNA-seq data of BM and spleen pDC of GSE114313  and bulk RNA-seq data of the splenic DC subsets pDC, cDC1 and cDC2 of GSE188992 .The question by the reviewer on the expression of the novel lncIrf8 is expressed in BM and spleen pDC. Expression of the cDC1 specific Tcons_00190258 lncRNA in BM and spleen pDC is very low to absent. The bulk RNA-seq data fully support these results: lncIrf8 and Tcons_00190258 lncRNA are expressed in pDC and cDC1 of spleen, respectively, and the expression pattern is the same as in in vitro bone marrow DC.The scRNA-seq data showed that .These new data are in new Figure 1—figure supplement 3 lncIrf8 across species. A lncRNA downstream of the human Irf8 gene was identified, referred to as LINC02132, but no lncRNA was observed downstream of zebrafish Irf8 gene  from BM cells of in vitro Flt3L cultures . In this study pDC were stimulated by CpG 2216 for 0, 2, 6 and 12 hours and subjected to RNA-seq analysis. Our analysis showed that Irf8 expression was upregulated with time upon CpG stimulation, while lncIrf8 expression was downregulated , including IRF8. CpG stimulation of pDC occurs within hours and is different from pDC differentiation, which takes a few days.This is an interesting question and we addressed this point by re-analyzing public RNA-seq data of FACS sorted pDC  these aspects could be studied without much effort as authors already have systems that can knockout lncIRF8 promoter or overexpress lncIRF8.lncIrf8 promoter KO severely compromises pDC development and pDC are essentially absent in lncIrf8 promoter KO cultures. (ii) lncIRF8 overexpression is difficult, as overexpression of lncRNA in general, which is challenging. This is because lncRNA overexpression in lentivirus vectors is very different from protein overexpression. To limit lncIrf8 overexpression to the lncIrf8 sequence a polyA signal for transcription termination has to be inserted at the 3 ’end of lncIrf8 . This polyA signal decrease the titer of lentivirus, which limits the overexpression capacity. We would expect to need a much higher lncIrf8 overexpression than the one achieved here  to reveal a potential significance of lncIrf8 in pDC functions, such as TLR stimulations and/or cytokine production.We understand the reviewer’s point on the interest in a potential significance of lncIRF8 on pDC functions using knockout or overexpression studies. However this point is difficult to address: (i) The lncIrf8 might have other roles in DC biology than the one identified in our study. We now included a sentence on the potential other roles of lncIrf8 in DC biology, which are not yet identified in the current study .We are in accord with the reviewer that 2. Though authors have identified that lncIRF8 is specifically expressed in pDC subtype but while understanding its significance and regulation, it was preferred to study bulk DC cultures overlooking the finding regarding specificity. Experiments performed by employing dCas9 VP64/KRAB-GFP constructs, do provide some direction in terms of mechanistic understanding of the cross-talk between the promoter of IRF8 and lncIRF8. This data needs to be further strengthened by sorting pDC, cDC1 and cDC2 populations and studying the effect of the genetic manipulations on IRF8 and lncRNA expression in each cell type.lncIrf8 and Irf8 promoters in sorted pDC, cDC1 and cDC2 populations. However, this is difficult and in some cases not possible, since the dCas9-VP64/KRAB-GFP constructs are introduced in HoxB8 multipotent progenitors (HoxB8 MPP)  and following Flt3L directed DC differentiation some DC subsets are very low in abundance to even absent . For example, in the dCas9-KRAB experiment in Figure 6A-E, frequencies of cDC1 and pDC are very low to absent. In the dCas9-VP64 experiment in Figure 5 cDC1 and pDC are there, but the impact of targeted CRISPR activation on the lncIrf8 promoter is mainly seen in HoxB8 MPP at day 0 of DC differentiation  and the activation effect is largely lost in the differentiated cells at later time points. Thus, studying the impact of targeted CRISPR activation and interference on IRF8 and lncRNA expression in DC subsets should include also MPP/CDP and pre-DC, and should be ideally performed by scRNA-seq and scATAC-seq. This analysis is beyond the scope of the present study and will be subject of our future work.We appreciate the reviewer’s suggestion on studying the effects of dCas9-VP64/KRAB-GFP constructs targeting the 3. One of the most important findings of the submitted study is to demonstrate that knocking out the lncIRF8 promoter also diminishes the expression of IRF8, pDC as well as cDC1 development . Once the IRF8 expression is suppressed the analysis of pDC and cDC1 differentiation is redundant. It is previously reported that the deletion of +32 Kb enhancer  in a mouse model did not affect pDC but specifically depleted cDC1 development. Authors should examine the deletion of only the +32 kb enhancer region in their culture system to sort out the hierarchy between the +32 Kb enhancer and +32 Kb lncRNA promoter in terms of DC subtype development (cDC1 Vs pDC). Does the deletion of the lncIRF8 promoter lead to epigenetic changes at the overall +32 region? It is expected that due to interactions and chromatin loop formation between IRF8 promoter with various enhancers elements; deletion of lncIRF8 promoter might play an important role in defining these interactions in a cell type-specific manner. These interactions become more valuable by the fact that activation of the lncIRF8 promoter leads to enhanced cDC1 differentiation. Does this simply mean more open chromatin conformation around the cDC1-specific +32 Kb region rather than correlating with the lncIRF8?lncIrf8 promoter might lead to epigenetic changes in the entire +32 region and might have multiple effects, such as affecting transcription factor binding, histone modifications, open chromatin regions and chromatin interactions. Similarly, targeting dCas9-VP64 to the lncIrf8 promoter for CRISPR activation caused more cDC1 differentiation, which might be also due to multiple effects, such as changes in transcription factor binding, chromatin interactions and/or chromatin conformation. Here CRISPR activation of the lncIrf8 promoter might cause +32 kb enhancer activation and as a consequence IRF8 activation and more cDC1 .We agree with the reviewer that deletion of the Nat. Immunol., 2019 by using the same gRNAs as described in Durai et al., in the following referred to as cDC1 specific +32 kb enhancer KO. In parallel, we also designed a novel pair of gRNAs to delete this +32 kb enhancer region. We followed the same procedure as the lncIrf8 promoter KO experiments  to obtain homozygous clones of cDC1 specific +32 kb enhancer KO. Five out of 165 single-cell clones were subjected to DC differentiation and further analyzed. cDC1 specific +32 kb enhancer KO did not affect pDC differentiation but compromised cDC1 development, which is in line with the phenotypes described by Durai et al., Nat. Immunol., 2019 and Murakami et al., Nat. Immunol., 2021 in mice.We now followed the request of the reviewer and deleted the +32 kb enhancer region with the four 4 AICE elements as described by Durai et al., These new results are now shown in new Figure 2—figure supplement 4 in the revised manuscript . The Materials and methods section was also updated accordingly . Please see above our response to Essential Revision 2.Can authors analyze the epigenetic changes by performing similar experiments in some of the available pDC and cDC1 cell lines? As these cell lines are likely to have active IRF8 transcription and it would help to measure the effect of lncIRF8 promoter deletion/mutation/activation/repression on IRF8 expression and specific DC subtype maintenance.We thank the reviewer for the suggestion to study the epigenetic changes reported here in some of the available pDC and cDC1 cell lines. However, we hesitate to do so, since established cell lines have inevitably suffered from mutation and multiple alterations, which are often unknown and/or hard to control. We rather have built our study on conditional immortalization of mouse bone marrow cells by HoxB8 .Eur. J. Immunol., 2022). Additionally, they stop proliferating when E2 is removed and faithfully recapitulate hematopoietic stem cell commitment, DC differentiation and allow genetic manipulations by CRISPR/Cas tools. Thus, we consider HoxB8 MPP a particularly versatile and useful model to study gene regulations during DC differentiation. Repeating the study in pDC and cDC1 cell lines will, in our opinion, only provide limited new information but entails the risk of picking up aberrant phenomena, which are inherent to studies in cell lines.These conditionally immortalized HoxB8 MPP exhibit an essentially unlimited lifespan when grown with estrogen (E2), when HoxB8 is active , which indicates that the gene regulation patterns we found in our in vitro systems also exist in vivo.We agree with the reviewer that validating the pDC and cDC1-specific interactions mediated by ex-vivo DC have a limited lifespan in vitro, thus it will be challenging to impossible to generate genetically modified cells for studying gene regulations in primary cells. In particular, deletions of cis-regulatory elements (such as enhancer or promoter sequences) that require single-cell clones, are not possible with primary mouse cells due to lifespan restrictions. These limitations made us to develop the conditional immortalized mouse bone marrow HoxB8 MPP system . Experimental details of the HoxB8 MPP-DC differentiation system are now included in a Guideline paper by Lutz et al., Eur. J. Immunol., 2022. We added this reference to the reference list and cite this paper along with the Xu et al., 2022 paper as indicated.Primary/5. Authors have also identified a TCONS_00190258 which is specifically expressed in the cDC1 subtype. Why not look at the expression levels of this transcript in the various experiments in the current study as enhancer element crosstalk will help in cell type-specific transcription of IRF8, lncIRF8, and TCONS_00190258?Tcons_00190258 is also express in ex-vivo tissue . Studying the impact of Tcons_00190258 on DC differentiation and its potential crosstalk with lncIrf8/Irf8 both in vitro and in vivo will be subject of our future work and described elsewhere.We really appreciate the suggestion made by the reviewer. We have now provided evidence that Reviewer #2 (Recommendations for the authors):I find the quality and interpretation of the genomics data to be excellent and I have no major concerns with that data. I do have some specific comments around the interpretation of the cellular data and the model presented.Specific comments1. The authors conclude that lncIRF8 promoter KO hematopoietic progenitors are not capable of differentiating to pDCs . However, it is now understood that Irf8 is not essential for pDC development (see Sichien Immunity 2016 where they study WT:IRF8-/- BM chimeras) and that Irf8 deregulates some key markers, including CD11b. The authors need to account for this discrepancy. Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO, and if not, why does this phenotype differ from the Irf8 KO?lncIrf8 promoter KO in vitro and following transplantation into sublethally irradiated mice in vivo, we observed low to absent CD11c+ CD11b- B220+ pDC  or Gr1- Siglec H+ pDC , respectively.We appreciate the point made by the reviewer. In our study on DC differentiation of Irf8-/- BM chimeras and the conclusions reached from their work that IRF8 is dispensable for pDC development. The results in Sichien et al., 2016 are based on WT:Irf8-/- BM chimeras upon transplantation in lethally irradiated mice and thus the divergent results compared to the classical Irf8-/- mice might be due to the experimental set-up WT:Irf8-/- BM chimeras versus Irf8 knockout mice .We know the work by Sichien et al., 2016 on WT:lncIrf8 promoter KO had an impact on DC marker genes, such as CD11c and CD11b. We visualized CD11c and CD11b expression (gating on single cells) on day 7 and 9 of Flt3L directed DC differentiation in lncIrf8 promoter KO and control cells. In lncIrf8 promoter KO cells CD11c expression was slightly reduced and CD11b expression was slightly increased .Thus, we propose the putative IRF8 repressor complex is only active in cDC and not pDC . We agree with the reviewer that at this stage of analysis we do not know why IRF8 binds to the 5’ part of the +32 kb enhancer in cDC but not in pDC. Addressing this point should be subject for further studies. See also our response above to Essential revisions 3.3. The authors use 2 versions of the HoxB8 MPP differentiation system (lines 207-234). Firstly, it is unclear, why two similar approaches are used, but more importantly, the second system outlined in figure 2 Suppl 3 shows that the lncIRF8 promoter is also required for cDC2 differentiation. This is a concern, as IRF8 is not expressed in cDC2, and Irf8-/- mice produce cDC2.lncIrf8 promoter KO cells, thereby following the DC differentiation systems described in Xu et al., Eur. J. Immunol., 2022:In the present study we used two approaches to differentiate the lncIrf8 promoter KO cells moved to Gr1+ monocytes . This spontaneous DC differentiation mimics in vitro the differentiation potential of these cells in vivo upon cell transplantation, where most of the lncIrf8 promoter KO cells also differentiated into Gr1+ monocytes .The first system is spontaneous DC differentiation, where we just remove estrogen (E2) and keep SCF, Flt3L (low concentration), hyper-IL6 and IGF1 in culture. Under these conditions most of the The second system is Flt3L directed DC differentiation, where estrogen (E2) and the growth promoting cytokines SCF, Flt3L (low concentration), hyper-IL6 and IGF1 are removed and high Flt3L concentration is applied to direct HoxB8 MPP differentiation towards DC, and all DC subsets  are obtained. In the manuscript we used both approaches, spontaneous DC differentiation (-E2) and Flt3L directed DC differentiation, to support the conclusions reached.lncIrf8 promoter KO most cells differentiated into Gr1+ monocytes  and thus CD11c+ cDC2 were much lower in the lncIrf8 promoter KO compared to control.Concerning the reduced cDC2 population in Figure 2—figure supplement 3G, this is because the cDC2 population was gated from CD11c+ cells and calculated based on living single cells. In the lncIrf8 promoter KO and control cells on day 8 and 10. On day 6 the spontaneous DC differentiation of control cells progressed slower than the lncIrf8 promoter KO cells, thus lower frequency of cDC2 was observed. We added this information to the legend for Figure 2—figure supplement 3 .We now also calculated cDC2 based on CD11c+ cells and show these data as new Figure 2—figure supplement 3H. We did not observe significant differences between


Vγ2 (Stargazin or TARPγ2) is a protein expressed in various types of neurons whose function was initially associated with a decrease in the functional expression of voltage-gated presynaptic Ca2+ channels (CaV) and which is now known to promote the trafficking of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) towards the cell membrane. Alterations in CaVγ2 expression has been associated with several neurological disorders, such as absence epilepsy. However, its regulation at the transcriptional level has not been intensively addressed. It has been reported that the promoter of the Cacng2 gene, encoding the rat CaVγ2, is bidirectional and regulates the transcription of a long non-coding RNA (lncRNA) in the antisense direction. Here, we investigate the proximal promoter region of the human CACNG2 gene in the antisense direction and show that this region includes two functional cAMP response elements that regulate the expression of a lncRNA called CACNG2-DT. The activity of these sites is significantly enhanced by forskolin, an adenylate cyclase activator, and inhibited by H89, a protein kinase A (PKA) antagonist. Therefore, this regulatory mechanism implies the activation of G protein-coupled receptors and downstream phosphorylation. Interestingly, we also found that the expression of CACNG2-DT may increase the levels of the CaVγ2 subunit. Together, these data provide novel information on the organization of the human CACNG2-DT gene promoter, describe modulatory domains and mechanisms that can mediate various regulatory inputs, and provide initial information on the molecular mechanisms that regulate the functional expression of the CaVγ2 protein.Ca Glutamate AMPA receptors (AMPAR) mediate much of the excitatory neurotransmission in the nervous system. The functional expression of AMPAR in the synapse is crucial in several forms of synaptic plasticity, including long-term potentiation and depression . TherefoFrom a molecular point of view, AMPA receptors are homo- or heteromeric complexes of ion-conducting subunits known as GluA1-4 that consist of four domains. The extracellular N-terminal domain, whose function is the assembly of the subunits and the location of receptors at the synapsis; the ligand binding domain, which undergoes conformational changes upon binding to glutamate that result in channel activation; the transmembrane domain, which forms the membrane’s ion channel and, the cytoplasmic C-terminal domain that regulates anchoring, trafficking, and cell signaling. Furthermore, native AMPARs are associated with auxiliary subunits such as the transmembrane AMPAR regulatory proteins (TARPs), which add functional diversity to the channels and contribute to determining their cellular localization .Vγ2, also known as Stargazin (Stg) or TARPγ2, is a multifunctional neuronal protein whose heterologous expression was initially shown to inhibit the functional expression of high voltage-activated calcium (CaV) channels  conformed by four transmembrane segments with amino and carboxyl termini located intracellularly. Notably, this protein is absent in the Stargazer mutant mouse, an animal model of epilepsy with absence seizures and cerebellar ataxia  domains and a Ca2+ regulatory element-binding factor (CaRE) element , and a TATA box that may regulate the transcription of a lncRNA called CACNG2-DT. Given the presence of functional CRE sites, it was reasonable to propose a possible modulation through the activation of a G protein-coupled receptor. It is well known that when this type of receptors is activated by its ligand, a nucleotide exchange occurs in the Gα subunit, and a conformational change is induced in the Gαβγ complex that results in its dissociation into two active signaling molecules, the Gα-GTP subunit and the Gβγ dimer . In sharp contrast, in the presence of H89, a PKA inhibitor, the transcriptional activity of the promoter is inhibited significantly as a consequence of a reduction in the levels of phosphorylated p-CREB.Here, we provide novel information on the bidirectional character of the human βγ dimer . The Gα-CACNG2-DT may increase the expression of CaVγ2/Stg, providing relevant information into the molecular mechanisms that regulate the expression of this intriguing synaptic protein.Finally, it is also known that lncRNAs are >200 bp RNA molecules that are not translated  and may CACNG2-DT gene promoter was determined by aligning a 1.3 kb 5′UTR region of the CACNG2 gene and the CACNG2-DT sequence deposited at NCBI (Gene ID: 105373021) using the Align Sequences Nucleotide BLAST software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The location of putative TSS was achieved using the Neural Network Promoter Prediction tool available on the Berkeley Drosophila Genome Project website (http://www.fruitfly.org/seq_tools/promoter.html). The presence of the TATA box and the possible binding sites for transcription factors and other relevant regulatory elements were determined using the Genomatix MatInspector tool (https://www.genomatix.de/). Finally, sequence analysis was performed with the Jaspar2022 tool (https://jaspar.genereg.net/) to identify possible CREB transcription factor binding sites.The sequence of the CACNG2-DT promoter constructs  and combinations of these (AB and BC) were obtained by PCR and cloned into the pGL3-Basic vector (Promega) (CACNG2-DT promoter in the NCBI database using the AlignX software from the Vector NTI Advance 11.5.0 bioinformatics package (Invitrogen). All constructs used in this study were automatically sequenced.Different Promega) . The conPromega) . The clo2. Cells were kept in culture to 80% confluence and transfected 48 h before being used, using the Lipofectamine 2000 Reagent according to the manufacturer’s instructions (Invitrogen) using 35 mm diameter culture dishes with 2 μg of each plasmid DNA encoding CaVγ2/Stg or CREB.Transfections were performed in the mouse neuroblastoma-derived cell line N1E-115 (ATCC). Cells were grown in DMEM medium, supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. Where indicated, experiments were performed in SH-SY5Y human neuroblastoma cells (ATCC) maintained in DMEM medium supplemented, as mentioned above. Both cell lines were kept in culture at 37°C in an atmosphere with 5% COCACNG2-DT gene cloned into the pGL3-Basic vector (2 µg) and cotransfected with the plasmid pRL-CMV as control (0.5 µg), using Lipofectamine and Reagent Plus. Five h later, the medium was replaced with medium supplemented with 250 µM adenosine . Forty-eight h after transfection, the promoter activity assays were performed using the Dual-Glo Luciferase Assay System (Promega) according to the suppliers’ instructions. Luminescence was measured using a luminometer (Modulus-Turner Biosystems). The firefly/Renilla luciferase activity ratio was used to normalize the data .In the case of experiments in which the endogenous adenosine receptor was activated, N1E-115 cells were transfected with the promoter region of the The activated CREB (p-CREB) levels were estimated by incubating the cells with 3 mM 8-Br-cAMP . Also, cells were transfected with a construct containing the human CREB1 coding sequence (pcDNA3-CREB1) to increase the amount of CREB. Likewise, the activation of the adelynate cylase was achived by incubating the cells with 10 μM Forskolin (Sigma-Aldrich). On the other hand, the decrease in p-CREB levels was also achieved by incubating the cells with 10 µM H89 (Sigma-Aldrich), a Protein Kinase A (PKA) inhibitor, or by silencing the transcription factor using small interference RNAs (siRNAs) .Photinus pyralis but lacks a promoter. The pRSV-βgal plasmid contains the coding sequence for β-galactosidase, which was used to normalize the results based on transfection efficiency. The Luciferase Assay System kit (Promega) was used for the assay. The CACNG2-DT promoter was also cloned into the pGL4Luc-RLuc vector (Addgene), which has the genes encoding the Photinus pyralis and Renilla reniformis luciferases separated by ∼100 bp, the region where the sequence of CACNG2-DT promoter was cloned. Hence, the synthesis of Photinus luciferase depended on the ability of the promoter to initiate transcription, while the expression of Renilla luciferase was associated with the antisense promoter transcriptional activity. The Dual-Glo Luciferase Assay System kit (Promega) was used for these assays.Constructs cloned into the pGL3-Basic vector were transfected in N1E-115 cells together with the pRSV-βgal plasmid (Promega). The pGL3-Basic vector contains the coding sequence for luciferase from Vγ2/Stg  or anti-β-actin  diluted in TBST. Next, membranes were washed with TBST and incubated with anti-mouse  or anti-rabbit  secondary antibodies coupled to horseradish peroxidase for 1 h. Finally, membranes were developed using the Immobilon Western reagent kit (Millipore). The chemiluminescence signal was detected with the Odyssey Fc kit (LI-COR).Cells were washed with PBS and incubated in lysis buffer (SDLB) containing 250 mM Tris-HCl [pH 8], 750 mM NaCl, 5% Triton x-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and the protease inhibitor cocktail Complete 1X (Roche diagnostics). Cells were then incubated at 4°C for 30 min, centrifuged, and the proteins in the supernatant quantified using the Protein Assay Dye Reagent Concentrate (Bio-Rad). Protein expression levels were determined by Western blot. To this end, 50 μg of proteins in Laemmli buffer were heated at 95°C for 5 min. The samples were loaded on a 10% SDS-PAGE gel, and the proteins were separated by electrophoresis using electrophoresis buffer . Next, the proteins were semi-dry transferred to nitrocellulose membranes. Membranes were washed with TBST buffer , and non-specific binding was blocked using TBST with milk (5%). Membranes were then incubated overnight with primary antibodies anti-CREB , anti-p-CREB , anti-CaCACNG2-DT promoter, using the 5′RACE System for Rapid Amplification of cDNA Ends kit (Invitrogen). Total RNA samples were isolated from SH-SY5Y cells and human cerebellum  with TRIzol Reagent (Ambion). The use of this tissue sample was a proved by the Biosafety Committee of the Center for Research and Advanced Studies of the National Polytechnic Institute (protocol No. 2023-1-CB). Oligonucleotides targeting the CACNG2-DT . Results are presented as mean ± SEM. Student’s t-test was used to compare two groups, while for the data analysis from several groups, ANOVA followed by Tukey’s Photinus pyralis firefly luciferase as a reporter gene, where the promoter of the CACNG2-DT gene was cloned. In parallel, the cells were cotransfected with an empty vector (without the promoter of interest) that only expresses the luciferase from sea pansy Renilla reniformis as a reporter gene, which was used to normalize the data and thus eliminate the transfection efficiency as a variable. The second system also used the very same construct that contains the Photinus luciferase as a reporter gene and the promoter of CACNG2-DT; however, in this case, the cells were cotransfected with an empty vector that expresses the enzyme β-galactosidase, which, as in the previous case, was used to normalize the data with the same purpose of eliminating transfection efficiency as a variable . To this end, we initially performed an in silico analysis to identify elements that could be regulating its activity. Next, the DNA of interest located between nucleotides 36702595 and 36703898 of the reference sequence in the NCBI database (RS_2023_03) was cloned into the pGL3-Basic vector. The cloned region is 1304 bp in size, 1158 corresponding to the 5′UTR region of the CACNG2 gene; an intergenic region of 123 bp, and a small sequence of 23 bp of exon 1. Interestingly, a TATA box located at position -1147 and -1133 was also found, as well as three nucleotides located at positions -1114, -978, and -150 that might function as TSS. Last, a CpG island, a common feature of bidirectional promoters, between nucleotides -643 and -278 was also identified   .CACNG2-DT gene promoter, the full-length construct cloned into the pGL3-Basic vector was transfected into N1E-115 cells treated with cAMP, and luciferase assays were performed. The results show that the CACNG2-DT promoter exhibited significantly higher transcriptional activity after cAMP treatment  designed for CREB silencing , an activator of adenylate cyclase, and H89, an inhibitor of PKA. The results showed that the cells treated with FSK (10 μM) have an increased capability to initiate transcription independently of the pcDNA3-CREB1 construct transfection . In contCACNG2-DT promoter.Likewise, in the case of the treatment with H89, the changes in the expression of CREB and p-CREB protein levels were confirmed by Western blot 48 h after transfection . Figure Next, given that the cells were transfected with the CREB construct, it proved interesting to observe changes in the CREB/p-CREB ratio and compare this ratio between the control and H89-treated groups. The results of this analysis are shown in in situ. Therefore, the activity of adenylate cyclase was next stimulated by activating a Gs-coupled receptor (adenosine) endogenously expressed in N1E-115 cells  is located at position -150 according to the CACNG2-DT exonic sequence, and proteins were extracted for Western blot analysis  the human CACNG2 promoter, like in the rat, is bidirectional and regulates the transcription of a lncRNA (CACNG2-DT) in the antisense direction; 2) the transcriptional activity of the gene promoter encoding CACNG2-DT may be greater than that of CACNG2; 3) the TSS is located at position +1 of the gene promoter encoding CACNG2-DT; 4) the cAMP-PKA/p-CREB signaling pathway is involved in the transcriptional regulation of CACNG2-DT; 5) the active form of CREB (p-CREB) could be regulating the activity of the gene promoter encoding CACNG2-DT through its binding to at least two CREB sites; and 6) CACNG2-DT may regulate the CaVγ2/Stg protein expression levels  REST construct has been used whose transfection into cell lines results in a marked increase in transcriptional activity, indicating a derepression of the promoter . Nevertheless, we could not confirm the presence of such a site experimentally. The reason for this discrepancy is also unknown; however, it may lie in the fact that this isoform might not be expressed in the cell line or the tissue used in our study. It is worth noting, however, that the TSS and the isoform mentioned above have only been found in silico. To our knowledge, no experimental evidence has been yet provided.On the other hand, in the rat genome, it has been reported that the d 578 bp . HoweverCACNG2-DT expression. The presence of functional CRE sites implies the activation of trimeric G protein-coupled membrane receptors and the subsequent activation of protein kinases that phosphorylate multiple targets.Likewise, transcriptional regulation through the binding of transcription factors to the cAMP response element (CRE) downstream of cAMP signaling plays important roles in many cellular processes. Transcription factors that bind to CRE, such as CRE-binding protein (CREB), are activated by cAMP-dependent PKA-mediated phosphorylation and stimulate gene expression by recruiting coactivators. Our study provides experimental evidence for the role of CREB in regulating CACNG2-DT could play a role in this process. On the other hand, investigating a potential association of these CREB elements with seizure phenotypes and pain-related plasticity in which CaVγ2/Stg has been implicated (It is worth mentioning here that CREB has many functions in different organs and tissues, including the brain, where its activity is related to learning and memory  where NMplicated  is also CACNG2-DT increases the CaVγ2/Stg protein levels, reinforcing the idea that the promoter of the CACNG2 gene is bidirectional and that one of its gene products is a lncRNA. This finding further provides experimental evidence for the regulated co-expression of the lncRNA-protein pair reported for other similarly organized genes (Another aspect to highlight has to do with the bidirectional nature of the promoter. It is well known that many promoters with this characteristic may give rise to a non-coding RNA and a protein-coding messenger . Interesed genes .CACNG2-DT increases the CaVγ2/Stg protein functional expression remains unclear, several possibilities exist to explain it. On the one hand, lncRNA could act in the nucleus regulating the transcriptional machinery. In addition, the CACNG2-DT could be involved in epigenetic regulation processes of gene transcription activation by modulating chromatin accessibility (CACNG2-DT could participate in diverse molecular processes, such as the modulation of signal translation pathways that favor the functional expression of the protein, the regulation of the mRNA’s stability and/or the regulation of the accessibility of miRNAs that control their expression (CACNG2-DT could interact with a miRNA that downregulates the functional expression of CaVγ2/Stg and, therefore, promotes an increase in protein levels. However, experimental evidence is still required to support this notion.Although the molecular mechanism by which sibility . On the pression . All thepression . In this