Thsd7a, is down-regulated without RORβ, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.Retinoic acid-related orphan receptor beta (RORβ) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORβ is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORβ delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORβ,  Localization of function is a fundamental principle organizing mammalian brain circuitry. Structure to function mapping is particularly striking in sensory input to L4 of the neocortex . L4 neurResearchers have long used the rodent whisker pathway to study cytoarchitecture development . The whiMany proteins and pathways are required for presynaptic organization of TCAs and/or postsynaptic organization in L4 . Much ofBarrel formation is also regulated transcriptionally. Transcription factors (TFs) such as Bhlhe22/Bhlhb5 and Eomes are involved in the early stages of cortical arealization and barrel development . DownstrThsd7a, that is down-regulated without RORβ and is required for maintained TCA organization in adulthood. Together these data characterize the role of RORβ across multiple levels to connect molecular and transcriptional mechanisms to cortical organization and place RORβ as a key regulator of a complex developmental transition orchestrating terminal L4 specification and initiating activity responsiveness.We show that in addition to being sufficient, RORβ is also required for both pre- and postsynaptic barrel organization. Without RORβ in the cortex, L4 neurons fail to migrate tangentially and do not organize into barrel wall structures. This also reduced TCA segregation shortly after barrel formation would have normally occurred. Interestingly, TCA segregation also declined as animals aged. Without RORβ, L4 gene expression and chromatin accessibility were disrupted, with L4-specific genes down-regulated and L5-specific genes up-regulated suggesting a disruption in terminal cellular identity. This involved complex changes in the expression and/or chromatin accessibility at binding motifs for many TFs in addition to RORβ, including developmental regulators and activity-regulated TFs. L4 neurons also received delayed excitatory input, a key step in barrel development. Lastly, we identify a putative direct gene target of RORβ, Cortical barrels in mice are complex structures. Cell-sparse barrel hollows are where thalamic projections are concentrated. Barrel walls are formed by cortical cell aggregates that surround the TCAs. Barrel septa consist of the intermediate spaces between barrel walls . To asseRorb locus. GFP/+Rorb mice express GFP in RORβ expressing cells allowing identification of barrel cortex without significant disruption to barrel structures or neuronal function (GFP/+Rorb mice were used as controls (Ctl), while GFP/GFPRorb mice disrupt both copies of Rorb to generate a KO. Controls showed no detectable disruption to barrel organization compared to WT animals , which contains a GFP expression cassette knocked-in to the function . RorbGFP animals  and 2A.Barrels form around postnatal day 5 . Nissl sRorb KO animals. Disorganization in the Rorb KO was characterized by both loss of quantifiable VGLUT2 contrast as well as the qualitative barrel patterning most obvious at P60 between Ctl and KO in While TCAs have been shown to instruct cortical cell organization we hypothesized the lack of barrel walls might reciprocally affect TCA organization. TCAs visualized by VGLUT2 staining showed an intact pattern of barrel hollows at P7 in KO animals, + inputs from outside the VPM we injected AAV expressing mCherry under the hSyn promoter specifically into the VPM  crossed to Cre driver lines generating tissue-specific disruption of RORβ as diagrammed in Cre from the serotonin transporter gene, Sert (Slc6a4 or 5-HTT) locus was used to knock-out Rorb in the thalamus. The CreSert line alone showed a mild disruption to TCA organization without disrupting barrel walls, suggesting the Cre knock-in might be hypomorphic (Rorb (Cre Rorbf/fSert) showed no additional disruption to TCAs or barrel walls. This is consistent with the observation that Rorb KO also did not disrupt barreloid organization . In addition, a CreCamK2a diver line that removes RORβ in the cortex after barrel formation, showed no effect. CreCamK2a activated expression of a tdTomato reporter from the Rosa26 locus in only a subset of GFP+ L4 neurons . At postnatal day 2 (P2) and prior to barrel formation, 246 genes were significantly disrupted with 51% down-regulated in the KO. At P7, just after barrel formation, 433 genes were disrupted with 36% down-regulated. At P30, 286 genes were disrupted with 37% down-regulated. Examining the overlap between ages we find very few genes significantly disrupted in the same direction across time points, suggesting highly dynamic and complex regulation, Because RORβ is a transcription factor we hypothesized loss of function would change gene expression in L4 neurons. To test this, RNA-seq was performed on sorted GFPRorb KO (DEGs). The Allen Brain Atlas was used to manually screen all DEGs for layer-specific expression in the neocortex. Genes were considered layer-specific if the in-situ hybridization (ISH) signal appeared at least three-fold higher in one layer (considering layers 2 and 3 together). Many genes had complex specificities showing enrichment in two or more layers. These were not included for simplicity. Grouping DEGs based on the layer they are normally expressed within, we see that DEGs which should be expressed in upper layers were generally down-regulated and DEGs that should be expressed in deep layers were generally up-regulated in the Rorb KO, Rorb, it is disproportionately weighted towards down-regulation of L4 genes and upregulation of L5 genes.RORβ expression is a key feature distinguishing L4 neurons . To examBcl11B  . To begis at P30 . High cos at P30 . DiffereWe hypothesized that many of the closed regions might contain a RORβ binding motif while regions that opened may have binding potential for other TFs. To assess this possibility, two software algorithms (MEME and HOMER) were used to identify de novo enriched motifs from the DNA sequences of differential ATAC peaks separating closed and opened regions. This unbiased analysis also identifies which enriched sequences match known TF binding motifs. RORβ was the top motif from closed regions, Rorb KO. In essence, we tested whether motifs were enriched around certain DEGs and whether a significant portion of those DEGs had a nearby motif. To reduce false positives, only motifs with significant enrichment in both tests are shown in To assess which TFs might play a significant role in up or down-regulation of DEGs we varied a distance window around the transcription start site (TSS) to identify nearby ATAC or control regions containing a DNA motif. We tested for enrichment of motifs in ATAC regions near DEGs compared to motifs in control regions. We also tested whether DEGs with a nearby motif were significantly enriched compared to a control group of genes that did not change expression in the Genes down-regulated at P30 showed significant enrichment of nearby RORβ motifs suggesting RORβ is important for gene activation . Motifs Rorb KO , cy at P7 , suggestl and KO . These dRorb KO and barrel organization, we examined known functions of genes differentially expressed at multiple developmental time points. Two candidates were identified with potential roles in cell migration and synaptogenesis. PlexinD1 (Plxnd1) is a cell signaling molecule known to play a role in pathfinding and synaptogenesis . Animals were provided with food and water ad libitum and kept on a 12 hr:12 hr light:dark cycle. Cages were enriched with huts, chew sticks, and tubes. All experiments were approved by the Institutional Animal Care and Use Committee of Brandeis University, Waltham, MA, USA.GFPRorb (1gRorb) and f/fRorb (flox/floxRorb) mice were obtained from Dr. Douglas Forrest ; floxplexinD1 ; Thrombospondin7a KO (Thsd7a) ; creEmx1 ; CreSert (Slc6a4) . creCamK2a . Forrest . RorbGFP isoform . The RorAnimals were fatally anesthetized and transcardially perfused with 15 mL 1x PBS  then 15 mL 4% PFA (Sigma Aldrich P6148-500G). Brains were fixed overnight in tangential orientation. After removing the whole brain from the skull, the cerebellum and olfactory bulbs were removed. The brain was split into two hemispheres along the longitudinal fissure and the midbrain was gently excised. The remaining cortex was placed in a shallow well made from a cryostat mold, filled with 4% PFA and a glass slide set on top for flattening. Brains were removed from PFA after 24–48 hr and stored in 30% sucrose/PBS solution at 4°C.AB_2665454) 1:500-1:1000, rabbit anti-VGLUT2  1:250, chicken anti-GFP  1:500-1:1000. Slices were washed three times in PBS for 10 min each at room temp and then moved to secondary antibody solution containing 0.3% Triton-X100, 3% Bovine Serum Albumin, 10% normal goat serum. All secondaries were used at 1:500; Goat Anti-Rabbit Alexa Fluor 564 , Goat Anti-Chicken Alexa Fluor 488 , Goat Anti-Rabbit Alexa Fluor 633 , Goat Anti-Guinea Pig Alexa Fluor 647 . Slices were stained using Nissl (Invitrogen N21479) at 1:250 in PBS for 2 hr at room temperature, washed in PBS as before, and mounted in VECTASHIELD HardSet Mounting Medium . Slides were stored at −20C and imaged within 1 week.50 µm slices were made on a freezing Microtome (Leica SM 2010R). Controls and KOs were stained together in batches. Slices were permeabilized overnight at 4°C in 0.3% Triton-X100  and 3% Bovine Serum Albumin (Sigma B4287-25G) in PBS. Slices were then incubated for 24 hr in primary antibody solution containing 0.3% Triton-X100% and 3% Bovine Serum Albumin (BSA) in PBS at 4°C. Primary antibody dilutions were as follows: Guinea pig anti-VGLUT2 . Contrast = (barrel - septa) / (barrel + septa). For absolute barrel or septa intensity, measurements were normalized to background regions  were drawn manually by a blinded researcher around 5–6 intact barrels from rows B, C, or D using Fiji . An ROI  regions  within eAddgene_114472) was delivered by stereotactic injection to the dorsal VPM of P18-20 animals. Mice were euthanized by cardiac perfusion of 4% paraformaldehyde solution at P30. Cortex was removed and flattened for tangential sectioning of barrel field into 50 µm slices on a freezing Microtome (Leica SM 2010R). Subcortical structures were embedded in agarose and sectioned into 50 µm coronal slices on a vibratome (Leica VT1000S), counterstained with DAPI, and imaged (Keyence BZ-X700). Barrel cortex was stained, imaged and contrast calculated as described above. We required two slices with a minimum of two mCherrry saturated barrels and no mCherry outside of the barrel field. Saturated barrels were defined as adjacent barrels surrounded by barrels with mCherry signal. Only saturated barrels were quantified and the same ROIs were used to quantify mCherry and VGLUT2.50 nl of AAV-hSyn-mCherry  or 484781 (Tox) also contained 10% NGS and 3% BSA to allow the probe binding step to also serve as the IHC blocking step. After developing the fluorescent in situ signal, slides were protected from light and stained overnight at room temperature with 1:250 chicken anti-GFP  diluted in 1X Tris-borate-EDTA (TBE) buffer containing 10% NGS and 3% BSA. Slides were washed four times in 1X TBE for 2–5 min and incubated for two hours at room temperature with 1:500 Goat Anti-Chicken Alexa Fluor 488 . Slides were washed four times in 1X TBE for 2–5 min, counterstained with DAPI and coverslips mounted according to the instructions for the RNAscope Fluorescent Multiplex kit. Batches of staining were balanced to contain equal numbers of control and Rorb KO samples per batch.Mice were euthanized by cardiac perfusion of 4% paraformaldehyde solution at P30. Brain tissue was pretreated according to the RNAscope Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue . Tissue was sectioned at 12 μm and subsequent staining performed according to the manufacturer’s instruction for the RNAscope Fluorescent Multiplex kit  with two protocol modifications. Antigen retrieval was carried out in an autoclave set to a 5 min ‘fast’ cycle, 121°C, 15 psi. After protease III digestion, probe solutions containing 313301-C2  using a rolling ball radius of 5 pixels. GFP signal was used to draw ROIs within L4 and L5. A custom CellProfiler  pipelineGFP/GFPRorb (KO) and GFP/+Rorb  mice were anesthetized with isoflurane and decapitated. Coronal slices (300 µm) containing the primary somatosensory cortex were cut on a Leica (VT1000S) vibratome and incubated at room temperature in ACSF containing (mM) 126 NaCl, 25 NaHCO3, 2.5 KCl, 1.2 NaHPO4, 2 CaCl2, 1 MgCl2 and 32.6 dextrose adjusted to 326 mOsm, pH 7.4 and saturated with 95%/5% O2/CO2. Submerged, whole cell recordings were performed at 32 ± 1° on an upright microscope (Olympus BX50) equipped with epifluorescence. Pipettes with resistance 4–6 Mohm were filled with internal solution containing (mM) 100 K-gluconate, 20 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP, 10 Na-phosphocreatine and 0.2% biocytin adjusted to 300 mOsm, pH 7.35. For mIPSC recordings, the internal included 133 mM KCl and gluconate was omitted to bring ECl to 0 mV. Recordings were made using an Axoclamp 700A amplifier, and were digitized at 10–20 kHz and analyzed using custom software running under Igor 6.03 (Wavemetrics). Miniature synaptic events were recorded in voltage clamp at −70 mV in the presence of PTX (mEPSCs) or DNQX+APV (mIPSCs) respectively.+ cell bodies within the GFP+ cell-dense layer 4. Input resistance was measured every 10–20 s with a small hyperpolarizing pulse and data were discarded if input or series resistance changed by >20%. P-values were calculated by 2-way ANOVA and adjusted for multiple comparisons by Tukey post hoc correction.Spiny stellate neurons were recognized based on their compact, GFP+ cells were isolated by FACS (BD FACSAria Flow Cytometer) from micro dissected L4 S1 live tissue . RNA-seq was performed as described previously . Briefly+ cells were isolated by FACS from microdissected L4 live tissue . The two independent samples were collected from a pool generated by combining tissue from two male and two female mice for a total of 8 animals used. Nuclei were transposed for 30 min and libraries amplified according to published methods (https://github.com/ENCODE-DCC/atac-seq-pipeline) was 34 ± 3 (mean ± SE). Peaks were identified permissively using HOMER (-style dnase –fdr 0.5 -minDist 150 -tbp 0 -size 75 -regionRes 0.75 -region) (ATAC-seq was performed as described previously . Briefly methods . Tagment methods  (https:/-region)  and IDR -region) . Differe-region) .Raw and processed RNA-seq and ATAC-seq files are available at GEO accession GSE138001.Rorb KO.Motifs identified de novo from the sequences underlying ATAC peaks was carried out using MEME AME with shuffled input sequences as control and default settings  , and HOM In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Thsd7a as a novel gene involved in barrel organization and also implicate several other genes as novel regulators of laminar identity. The observation that barrel organization declines with age in wildtype mice is an interesting one that provides new avenues of research. Thus, overall, this work represents a significant contribution to our understanding of a complex cortical developmental process.This study provides insights into the molecular mechanisms underlying RORβ's involvement in barrel formation and thus provides a tantalizing link across gene expression, chromatin accessibility, excitatory activity, and cellular organization in L4. The data highlight Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Catherine Dulac as the Senior Editor.Thank you for sending your article entitled "Cortical RORβ is required for layer 4 transcriptional identity and barrel integrity" for peer review at Thsd7 knockout. The reviewers raised concerns about interpretation of the data supporting each of these findings, and felt that additional experiments would be required to support either claim. These concerns are most clearly articulated in the comments of reviewer #3 below but were voiced by all three reviewers during the discussion period.The three reviewers were split on the significance of the current study as written but all felt there were two findings in particular that, if they could be strengthened, would be of broad interest. These points were 1) the layer 4 to layer 5 "identity switch" of the RORb knockout neurons and 2) the degeneration of the organization of thalamocortical axons with age and/or Essential revisions:1) Alternative methods in addition to FACS purification by GFP and sequencing are needed to confirm that the layer 5 gene expression programs are indeed occurring in what would have been layer 4 RORb+ neurons.Thsd7 knockout mice to confirm that this organization are disrupted.2) Complementary methods in addition to vGLut2 staining are needed to characterize the arborization of TC axons in aging and in the Reviewer #1:Thsd7a, which also seems to disrupt some aspects of barrels.This manuscript addresses the circuit functions of the layer 4 neuron marker RORb. The premise of the study is that layer 4 neurons are somewhat unique in their morphological arrangement into sensory input defined cellular aggregates and the authors presuppose that there is a transcriptional basis for this. Prior studies support this idea and already a number of transcription factors are known that link thalamocortical inputs with the organization of sensory barrels. Here the authors add RORb to this list, knocking it out and showing disruption of barrel formation. Global knockout of RORb was shown to reduce barrel formation (though barrels are still visible). Conditional knockout showed that RORb was required in the cortex (Emx1-cre) prior to barrel formation to have this effect. In both cases the effects get worse with age, though RORb knockout by CamKII-cre, after the barrels have formed, appeared to have no effect. The authors took advantage of the fact that the constitutive knockout allele expresses GFP to purify cells from S1. They compared RNA-seq data against Allen Brain in situ data for layer 4 markers and saw an apparently transformation of the cell fate in the KO neurons, with a down regulation of layer 4 markers and upregulation of layer 5. Using ATAC-seq to find the transcriptional mechanisms of these changes the authors found RORb motifs in many differentially-accessible regions comparing WT and KO, though not near the layer 4 genes. Many other sites contained binding sites for activity-sensing TFs, consistent with the known role of sensory input in barrel development. Finally the authors explore one potential target, The work is well done, the studies are rigorous, and the manuscript is well written. However it is not clear that the story is highly novel or significant. Several TFs (as the authors cite) are already known to couple barrel development with sensory input. The authors find some interesting information about RORb's specific functions in this process, but it is not clear that fundamental new insights emerge from the data. Overall I see the manuscript as being of great interest to specialists, but of limited interest to the broader audience.Reviewer #2:Thsd7a as a novel gene involved in barrel organization and also implicate several other genes as novel regulators of laminar identity. The observation that barrel organization declines with age in wildtype mice is an interesting one that provides new avenues of research. Thus, overall, this work represents a significant contribution to our understanding of a complex cortical developmental process. The body of the manuscript is well written, though I would urge the authors to carefully review the figure legends which contain errors and require some editing. In general, the conclusions drawn by the authors are supported by the data. The tests chosen for statistical analysis seem appropriate and sufficiently rigorous.This study provides insights into the molecular mechanisms underlying RORβ's involvement in barrel formation and thus provides a tantalizing link across gene expression, chromatin accessibility, excitatory activity, and cellular organization in L4. The data highlight 1) It would have been nice to see the progression from the molecular to the cellular level extended to the functional level to complete the whole picture, for instance by using a behavioral test of whisker function to see whether these molecular and cellular changes translate to a meaningful functional phenotype.2) The mini EPSC frequency appears to stabilize in Rorb KO mice by P10, yet at P30 the authors observed changes in the expression of many activity-dependent TFs. To strengthen the claim that the changes in excitatory activity are consistent with the observed transcriptional changes, it would seem appropriate to perform the mini EPSC experiment at the P30 time point when most of the activity-dependent TFs show up-regulated expression.Thsd7a experiment . It seems prudent to use this approach in all the other experiments .3) It is unclear why normalization was only used when quantifying changes in barrel-septa contrast in the 4) Figure 3D lacks an appropriate negative control for comparison at P60 . This is especially important since the authors demonstrate an age-related desegregation of TCA.Reviewer #3:Using conditional KO strategies authors show that the transcription factor RORb is required in the cortex but not in thalamus for barrel formation; then, based on extensive genomic analyses of RORb+ neurons from layer 4 (L4) neurons , they find that RORb+ neurons loose some L4 molecular characteristics while acquiring new L5 characteristics. Finally they analyse 2 target genes of RORb looking for morphological change in barrel organisation.Overall these data are interesting and well analysed; on one hand they confirm the role of RORb in L4 barrel formation ; this has had been proposed based on expression data and on gain of function experiments . On the other hand, they bring an interesting controversy on the role of RORb in acquiring L4 molecular identity. L4 is generally considered to be part of the upper cortical plate neurons, and to share developmental origin and molecular identity with L2/L3 neurons .The following weak points need however to be addressed.Rorb-KO. However, this based solely on changes in gene expression, which are not really compelling, because effects are not consistent and vary strongly with age. To support their conclusion, authors need to provide further evidence on the laminar distribution of L4 and L5 /L6 molecular markers . Additionally it would be important to know whether L4 neurons acquire new morphological characteristics, of L5 neurons such as pyramidal shape and sub-cerebral projections. Because GFP is expressed in the RORb deficient neurons these could be easily traced and analyzed.1) The most original claim of the study is that L4 neurons acquire L5 identity in 2) The changes in gene expression in RORb KO vary most between P2 and P7: L5 markers (e.g. ctip2 and Fez1) are up regulated only at P2. Could there be some contamination of the P2 samples with L5 , despite efforts in the dissection?+ cortical inputs than TCAs. In fact the difference between WT and RORb- KO becomes is less clear as animals age  and authors note "loss of RORb did not significantly change the time course of TCA desegregation”. Thus without complementary approaches it is hard to make such strong conclusions. Previous studies showing desegregation of TCAs in the barrel cortex, secondary to cortex-specific deletions have used complementary methods such as tracing reconstructions of TCA axon terminals in the cortex .3) Another strong claim is that segregation of TCAs degrades with age. However, this is based exclusively on Vglut2 immunostaining with low resolution. This is questionable as the cortex matures, since Vglut2 staining becomes much more diffuse, possibly because of the arrival of other VGluT2Thsd7-KO is not really convincing. Although some higher resolution images are shown, these are not confocal, and would not allow rigorous measures of VGluT2+ terminals.4) For similar reasons as above, the phenotype of the barrel phenotype of Rorb is required for layer 4 transcriptional identity and barrel integrity" for consideration by eLife. Your revised article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Catherine Dulac as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Patricia Gaspar (Reviewer #3).Thank you for resubmitting your article "Cortical The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Thsd7a as a novel gene involved in barrel organization and also implicate several other genes as novel regulators of laminar identity. The observation that barrel organization declines with age in wildtype mice is an interesting one that provides new avenues of research. Thus, overall, this work represents a significant contribution to our understanding of a complex cortical developmental process.This study provides insights into the molecular mechanisms underlying RORβ's involvement in barrel formation and thus provides a tantalizing link across gene expression, chromatin accessibility, excitatory activity, and cellular organization in L4. The data highlight Essential revisions:This is a revised manuscript, and in a previous round of review the reviewers requested a plan from the authors outlining how they would address what were seen as two major concerns. These were the claim from RNA-seq data that Layer 4 neurons were expressing Layer 5 gene programs, and the use of vGlut2 labeling to assess inputs to barrel cortex. The authors proposed to offer new data to address each of these points, using quantitative in situ to validate the RNA-seq and a viral tracing method to complement the vGLut2 staining evaluation of the organization of inputs in barrel cortex.The authors did provide these data, however two of the reviewers felt that these new figures needed more clarification. In particular the way the authors presented and interpreted the viral input labeling experiment was a source of significant confusion. Comments from the reviewers on these two new datasets are below and these need to be addressed with text/figure revisions and/or tempering of claims.Barrel cortex viral input labeling:– The fact that the TC barrelless phenotype increases with age is not supported by the additional evidence provided. Furthermore, the quantification in Figure 2B actually shows the opposite trend: using measures of VMAT2 intensity in barrel hollows/septae the Ctrl/KO difference is most marked at P7 and P20, it seems less marked at P30 and it is no longer significant at P60. Therefore I would suggest not highlighting this in the Abstract and Discussion without better evidence.– Figure 2—figure supplement 1 and Figure 7—figure supplement 1, compare TC tracing (very nice) with Vglut2 to show this is similar, but they do not illustrate/compare data from the ctrl and KO on the same or graph. The mean control values (n = ?) are represented by the dashed line; but then the SEM and stats need to be added. These 2 figures could be combined into one clearer figure.– I do not understand the authors' interpretation of Figure 2—figure supplement 1C and Figure 7—figure supplement 1. First, what is the y-axis in Figure 2—figure supplement 1C and Figure 7—figure supplement 1 – barrel septal contrast of what? VGlut2? Comparing the absolute contrast levels of two methods of labeling inputs at a single time point or in a single genotype would not seem to mean much. I was assuming when they suggested this method that the authors would compare the Cherry signal in WT and KO to show it reproduced the lower signaling in the KO relative to WT like they saw with vGLut 2. I thought they would do a similar experiment and look at cherry labeling over time in the WT to show it decreases similar to the vGlut2 labeling. Those comparisons would seem to have been needed to address the concerns that were raised by the reviewers about the vGlut2 signal.RNAscope in situ quantification:– What is the time point for the in situ in Figure 5A? Why is the difference in Fezf2 expression so large in this sample whereas it is negligible at all time points in Supplementary Figure 4B? What did Tox look like in the sequencing data – it would be good to include it in Supplementary Figure 4B. The authors should also show the images for Tox in the supplementary figure because this is important to the validation. Finally, I do not understand what Figure 4E is trying to show or what the authors are concluding. There is no quantification so it is not possible to judge whatever conclusion was intended. Essential revisions:1) Alternative methods in addition to FACS purification by GFP and sequencing are needed to confirm that the layer 5 gene expression programs are indeed occurring in what would have been layer 4 RORb+ neurons.2) Complementary methods in addition to vGLut2 staining are needed to characterize the arborization of TC axons in aging and in the Thsd7 knockout mice to confirm that this organization are disrupted.Reviewer #1:This manuscript addresses the circuit functions of the layer 4 neuron marker Rorb. The premise of the study is that layer 4 neurons are somewhat unique in their morphological arrangement into sensory input defined cellular aggregates and the authors presuppose that there is a transcriptional basis for this. Prior studies support this idea and already a number of transcription factors are known that link thalamocortical inputs with the organization of sensory barrels. Here the authors add RORb to this list, knocking it out and showing disruption of barrel formation. Global knockout of RORb was shown to reduce barrel formation (though barrels are still visible). Conditional knockout showed that RORb was required in the cortex (Emx1-cre) prior to barrel formation to have this effect. In both cases the effects get worse with age, though RORb knockout by CamKII-cre, after the barrels have formed, appeared to have no effect. The authors took advantage of the fact that the constitutive knockout allele expresses GFP to purify cells from S1. They compared RNA-seq data against Allen Brain in situ data for layer 4 markers and saw an apparently transformation of the cell fate in the KO neurons, with a down regulation of layer 4 markers and upregulation of layer 5. Using ATAC-seq to find the transcriptional mechanisms of these changes the authors found RORb motifs in many differentially-accessible regions comparing WT and KO, though not near the layer 4 genes. Many other sites contained binding sites for activity-sensing TFs, consistent with the known role of sensory input in barrel development. Finally the authors explore one potential target, Thsd7a, which also seems to disrupt some aspects of barrels.The work is well done, the studies are rigorous, and the manuscript is well written. However it is not clear that the story is highly novel or significant. Several TFs (as the authors cite) are already known to couple barrel development with sensory input. The authors find some interesting information about RORb's specific functions in this process, but it is not clear that fundamental new insights emerge from the data. Overall I see the manuscript as being of great interest to specialists, but of limited interest to the broader audience.Thsd7a KO, two findings further supported by additional VPM-specific experiments, opens up the barrel cortex to study mechanisms involved in age-related plasticity and cytoarchitecture maintenance.Several features of our study make it of broad interest. Prior studies identified transcription factors required for barrel development and laminar fate but stopped short of revealing the underlying transcriptional networks. Here, we begin to open the black box by which loss of a single transcription factor, through a cascade of changes in gene expression and chromatin accessibility, can shape activity-dependent cortical development. Our study dives deeper into the transcriptional regulation of barrel development than previous studies. The mechanisms we highlight have broader implications for understanding the complexity of transcriptional networks governing cellular identity and the diversity of transcriptional mechanisms altered by a single TF. Additionally, our description of TCA desegregation in adulthood and the increase in adult desegregation after Reviewer #2:This study provides insights into the molecular mechanisms underlying RORB's involvement in barrel formation and thus provides a tantalizing link across gene expression, chromatin accessibility, excitatory activity, and cellular organization in L4. The data highlight Thsd7a as a novel gene involved in barrel organization and also implicate several other genes as novel regulators of laminar identity. The observation that barrel organization declines with age in wildtype mice is an interesting one that provides new avenues of research. Thus, overall, this work represents a significant contribution to our understanding of a complex cortical developmental process. The body of the manuscript is well written, though I would urge the authors to carefully review the figure legends which contain errors and require some editing. In general, the conclusions drawn by the authors are supported by the data. The tests chosen for statistical analysis seem appropriate and sufficiently rigorous.1) It would have been nice to see the progression from the molecular to the cellular level extended to the functional level to complete the whole picture, for instance by using a behavioral test of whisker function to see whether these molecular and cellular changes translate to a meaningful functional phenotype.While we agree extending the findings into behavioral readouts would be very interesting, those experiments are very time and resource intensive. We feel they are beyond the scope of this paper which focuses on molecular transcriptional mechanisms, and their roles in anatomical and physiological development.2) The mini EPSC frequency appears to stabilize in Rorb KO mice by P10, yet at P30 the authors observed changes in the expression of many activity-dependent TFs. To strengthen the claim that the changes in excitatory activity are consistent with the observed transcriptional changes, it would seem appropriate to perform the mini EPSC experiment at the P30 time point when most of the activity-dependent TFs show up-regulated expression.We previously performed mEPSC recordings at P19 and found that both frequency and amplitude returned to normal levels. We have added this data to Figure 6B.3) It is unclear why normalization was only used when quantifying changes in barrel-septa contrast in the Thsd7a experiment . It seems prudent to use this approach in all the other experiments .contrast calculation. It was used to measure absolute intensity within barrels or septa separately in Figure 7E-F. Because the contrast value is a ratio, it does not need to be normalized as both the barrels and septa would be corrected by the same measurement. We have added more detail regarding this aspect to the Materials and methods (subsection “Imaging and fluorescence quantification”).Normalization was not used in Figure 7D in the barrel-septa 4) Figure 3D lacks an appropriate negative control for comparison at P60 . This is especially important since the authors demonstrate an age-related desegregation of TCA.We are not able to detect a significant difference in barrel contrast between P30 and P60 in our control data . There is a slight downward trend but the variance is too high and our N is too small to determine whether this is significant at the thresholds we’ve set. We do detect a significant change in TCA segregation comparing P7 to P20. We have added a more detailed description to the Results to make this distinction clearer ”).f/fRorb CamK2a-cre does not affect TCA organization, it is very unlikely that the CamK2a-cre alone will have an effect. We do not think excluding this control changes the interpretation of the negative result.Given that the Reviewer #3:Using conditional KO strategies authors show that the transcription factor RORb is required in the cortex but not in thalamus for barrel formation; then, based on extensive genomic analyses of RORb+ neurons from layer 4 (L4) neurons , they find that RORb+ neurons loose some L4 molecular characteristics while acquiring new L5 characteristics. Finally they analyse 2 target genes of RORb looking for morphological change in barrel organisation.Overall these data are interesting and well analysed; on one hand they confirm the role of RORb in L4 barrel formation ; this has had been proposed based on expression data and on gain of function experiments . On the other hand, they bring an interesting controversy on the role of RORb in acquiring L4 molecular identity. L4 is generally considered to be part of the upper cortical plate neurons, and to share developmental origin and molecular identity with L2/L3 neurons .The following weak points need however to be addressed.1) The most original claim of the study is that L4 neurons acquire L5 identity in RORb-KO. However, this based solely on changes in gene expression, which are not really compelling, because effects are not consistent and vary strongly with age. To support their conclusion, authors need to provide further evidence on the laminar distribution of L4 and L5 /L6 molecular markers . Additionally it would be important to know whether L4 neurons acquire new morphological characteristics, of L5 neurons such as pyramidal shape and sub-cerebral projections. Because GFP is expressed in the RORb deficient neurons these could be easily traced and analyzed.We note that we are not claiming that loss of RORβ leads to a fate-switch in which all aspects of L4 identity are lost and all aspects of L5 identity are gained. Instead, we believe the data argue for a more nuanced view of “identity” comprised of multiple transcriptional circuits, only some of which are disrupted by loss of RORβ. This issue is likely in part due to a lack of clarity in our description. Our use of the phrase “shift in identity” understandably, but also unintentionally, evokes the concept of fate switching. We have edited the manuscript to make it clear  that we don’t think loss of RORb causes L4 neurons to take on a L5 identity. We have also added an analysis of additional layer-specific genes identified from the Allen Brain Atlas demonstrating that while many L4 and L5 genes are dysregulated, many are not . Additionally, we don’t find any strong evidence that the L4 neurons have acquired L5 morphology. Instead, we propose that loss of RORb disrupts L4 specification, a process which appears to involve repression of L5 genes. In fact, we think RORb’s role is more likely in fine-tuning L4 identity after fate selection, and upregulation of deeper layer genes is a symptom of dysregulated L4 specification rather than a large-scale identity switch. We apologize for our lack of thoughtfulness in word choice and phrasing while describing these transcriptional changes.2) The changes in gene expression in RORb KO vary most between P2 and P7: L5 markers (e.g. ctip2 and Fez1) are up regulated only at P2. Could there be some contamination of the P2 samples with L5 , despite efforts in the dissection?While we agree with reviewer #3 that age is a factor in upregulation of L5 genes, we do not agree with the assessment that P2 and P7 showed the most change between control and KO. The red line graphs of Figure 4B show that the younger ages show more variation in L5 genes changes with several genes down regulated at younger ages but ultimately upregulated at P30. The P30 adult time point shows the most consistent upregulation with the bulk of the L5 DEGs increased, and for several genes, also with the largest fold increase. We have added a line showing the mean LFC across the group of genes in Figure 4B to make this conclusion easier to assess. While Ctip2 does show the strongest upregulation at P2, FezF2 (Fez1 is not a DEG in our dataset) is only upregulated at P30.Rorb KO. This would be unexpected if the changes seen in the genes shown in Figure 4 were due to contamination.The reviewer is insightful to suggest possible difficulty in the P2 microdissection. While we think the most compelling RNA-seq evidence is at P7 and P30 we performed RNAscope in situ hybridization to confirm upregulation of two L5 genes, FezF2 and Tox . As an additional control, we analyzed expression changes in all of the genes differentially expressed between layers 4 and 5 of somatosensory cortex according to the Allen Brain Atlas . Most genes (74 L4 and 190 L5) did not, on average show a change between WT and + cortical inputs than TCAs. In fact the difference between WT and RORb- KO becomes is less clear as animals age  and authors note "loss of RORb did not significantly change the time course of TCA desegregation”. Thus without complementary approaches it is hard to make such strong conclusions. Previous studies showing desegregation of TCAs in the barrel cortex, secondary to cortex-specific deletions have used complementary methods such as tracing reconstructions of TCA axon terminals in the cortex .3) Another strong claim is that segregation of TCAs degrades with age. However, this is based exclusively on Vglut2 immunostaining with low resolution. This is questionable as the cortex matures, since Vglut2 staining becomes much more diffuse, possibly because of the arrival of other VGluT2+ terminals.4) For similar reasons as above, the phenotype of the barrel phenotype of Thsd7-KO is not really convincing. Although some higher resolution images are shown, these are not confocal, and would not allow rigorous measures of VGluT2Thsd7a, rather than as a result of the arrival of other VGLUT2+ cortical inputs as the cortex matures.To complement our VGLUT2 staining quantification and to address comments 3 and 4, we injected AAV carrying an mCherry reporter gene driven by the hsyn promoter (AAV-hSyn-mCherry) into VPM to anatomically label TCAs. We use VLGUT2 staining to define barrels and compared the barrel-septa contrast calculated from mCherry expression in VPM axons to that calculated from VGLUT2. Figure 2—figure supplement 1A-C and Figure 7—figure supplement 1 show that the contrast calculated from VPM-specific mCherry expressing axons was similar to the contrast calculated from VGLUT2 staining. This supports our interpretation that VPM TCA organization degrades with age and without functional Essential revisions:This is a revised manuscript, and in a previous round of review the reviewers requested a plan from the authors outlining how they would address what were seen as two major concerns. These were the claim from RNA-seq data that Layer 4 neurons were expressing Layer 5 gene programs, and the use of vGlut2 labeling to assess inputs to barrel cortex. The authors proposed to offer new data to address each of these points, using quantitative in situ to validate the RNA-seq and a viral tracing method to complement the vGLut2 staining evaluation of the organization of inputs in barrel cortex.The authors did provide these data, however two of the reviewers felt that these new figures needed more clarification. In particular the way the authors presented and interpreted the viral input labeling experiment was a source of significant confusion. Comments from the reviewers on these two new datasets are below and these need to be addressed with text/figure revisions and/or tempering of claims.Barrel cortex viral input labeling:– The fact that the TC barrelless phenotype increases with age is not supported by the additional evidence provided. Furthermore, the quantification in Figure 2B actually shows the opposite trend: using measures of VMAT2 intensity in barrel hollows/septae the Ctrl/KO difference is most marked at P7 and P20, it seems less marked at P30 and it is no longer significant at P60. Therefore I would suggest not highlighting this in the Abstract and Discussion without better evidence.The paper does not argue that the effect of Rorb KO increases with age. Instead, we find that segregation of afferents declines with age and this effect is separate from the more profound loss of segregation seen in the knockout. This was highlighted by reviewer #2 in the prior set of reviews: “The observation that barrel organization declines with age in wildtype mice is an interesting one that provides new avenues of research. Thus, overall, this work represents a significant contribution to our understanding of a complex cortical developmental process.” To try to make this point clearer, we have altered the sentence in the Abstract which previously read “Interestingly, barrel organization also degrades with age.” to read: “Interestingly, barrel organization also degrades with age in wildtype mice.” We have also clarified this portion of the Discussion by changing: “Our observation that barrel organization declined with age is very interesting and possibly the first description of this phenomenon in mice ” to “Our observation that barrel organization declined with age in wildtype animals is very interesting and possibly the first description of this phenomenon in mice .” Please also note that we are careful to state in the Results that the change with age is common to both control and KO animals and that “while both age and loss of RORβ significantly reduced contrast, loss of RORβ did not significantly change the time course of TCA desegregation”.– Figure 2—figure supplement 1 and Figure 7—figure supplement 1, compare TC tracing (very nice) with Vglut2 to show this is similar, but they do not illustrate/compare data from the ctrl and KO on the same or graph. The mean control values (n = ?) are represented by the dashed line; but then the SEM and stats need to be added. These 2 figures could be combined into one clearer figure.The prior round of reviews included the following request from reviewer #3:+ cortical inputs than TCAs…4) For similar reasons as above, the phenotype of the barrel phenotype of Thsd7-KO is not really convincing.”“3) Another strong claim is that segregation of TCAs degrades with age. However, this is based exclusively on Vglut2 immunostaining with low resolution. This is questionable as the cortex matures, since Vglut2 staining becomes much more diffuse, possibly because of the arrival of other VGluT2Thsd7-KO. Specifically, in the prior round of reviews Essential revision #2 reads “Complementary methods in addition to vGLut2 staining are needed to characterize the arborization of TC axons in aging and in the Thsd7 knockout mice to confirm that this organization are disrupted.”I realize now that the reviewer may have thought that we were claiming that the Rorb KO phenotype increases with age (see point immediately above) but at the time we and the editors clearly interpreted the reviewer’s comments as referring solely to the aging phenotype, which is also present in wildtype mice, and the Based on our understanding of these requests we submitted a revision plan that included the a response as follows:“To complement our vGLUT2 staining quantification we propose to inject anterograde tracers into the VPM of adult wild-type and Ths7a KO animals. We will then quantify the barrel-septa contrast both of vGLUT2 staining and the axonal label. We will also use high resolution confocal microscopy to document the presence of axonal terminals in the septa. If the loss of vGLUT2 contrast in the older adults and Ths7a KO is primarily due to loss of TCA localization to barrel hollows we should see a comparable loss of contrast in the VPM anterograde tracer. On the other hand, if the loss of vGLUT2 contrast is due to ingrowth of other vGluT2+ afferents, the TCA and vGLUT2 contrast will be mismatched and axonal labeling in the septa will be absent.”Rorb KO animals to wildtype animals or to produce a complete time course. While we did not produce a full time course for Figure 2—figure supplement 1, we replot the Vglut2 contrasts obtained for P7 for reference and for P30 to show that both measures of Vglut2 contrast are similar despite the experiments having been carried out many months apart. We felt it was important to show that the new experiments involving surgical and viral methods did not significantly alter Vglut2 contrast. The key result shown by these experiments is that TCAs specifically originating in the VPM do not show a difference in contrast compared to total Vglut2 staining. This data rules out the alternative interpretation of non-thalamic afferents raised by the reviewer in the first round of comments. If loss of Vglut2 contrast was due to the arrival of other VGluT2+ inputs we would not see such close alignment of Vlglut2 contrast and mCherry contrast at P30.We did carry out these experiments and illustrate them in Figure 2—figure supplement 1 and Figure 8—figure supplement 1. The number of animals for these experiments (4 for both figures) is stated in the legends. We did not propose to directly compare As the reviewer suggests, we have restated the N’s for the control data in the legends and have given the statistics in the two legends as well. We have also added the SD to the figures as requested.Ths7a KO.We do not believe it would be clearer to combine the two figures into one since they come at very different points within the manuscript and address different questions. Figure 2—figure supplement 1 addresses the decline of barrel segregation with age in normal mice, while Figure 8—figure supplement 1 addresses the loss of barrel-septa contrast in the – I do not understand the authors' interpretation of Figure 2—figure supplement 1C and Figure 7—figure supplement 1. First, what is the y-axis in Figure 2—figure supplement 1C and Figure 7—figure supplement 1 – barrel septal contrast of what? VGlut2? Comparing the absolute contrast levels of two methods of labeling inputs at a single time point or in a single genotype would not seem to mean much. I was assuming when they suggested this method that the authors would compare the Cherry signal in WT and KO to show it reproduced the lower signaling in the KO relative to WT like they saw with vGLut 2. I thought they would do a similar experiment and look at cherry labeling over time in the WT to show it decreases similar to the vGlut2 labeling. Those comparisons would seem to have been needed to address the concerns that were raised by the reviewers about the vGlut2 signal.+ cortical inputs than TCAs” can be ruled out.The y-axis in both figures compares the contrast of VGlut2 to the contrast of mCherry contained within VPM axons. Unlike intensity, contrast is not an absolute measure, but a relative one. It measures the fractional difference between labeling within the barrel compartment relative to that within the septal compartment as a measure of the segregation of axons. A direct comparison of the spatial contrast (segregation) between Vglut2 labelled axons and mCherry-labelled axons in the same tissue was precisely the point of this experiment. What this figure shows clearly is that both methods of labeling axons produce a nearly identical measure of contrast. In other words, the original concern that “Vglut2 staining becomes much more diffuse, possibly because of the arrival of other VGluT2RNAscope in situ quantification:– What is the time point for the in situ in Figure 5A?The in situ was performed at P30 which is stated in the Materials and methods. We have now added this to the legend for clarity.Why is the difference in Fezf2 expression so large in this sample whereas it is negligible at all time points in Supplementary Figure 4B?The scale in Supplementary Figure 4B made the difference appear negligible because Fezf2 levels decline greatly by P30. We have replotted the data from Supplementary Figure 4B so that the close agreement between the sequencing and in situ can be better appreciated. We have also included this panel in a new main figure  as suggested below.What did Tox look like in the sequencing data – it would be good to include it in Supplementary Figure 4B. The authors should also show the images for Tox in the supplementary figure because this is important to the validation.Tox and sequencing data for Tox to allow appreciation of the correspondence. This now comprises a new Figure 5—figure supplement 1.We have now included both images for Finally, I do not understand what Supplementary Figure 4E is trying to show or what the authors are concluding. There is no quantification so it is not possible to judge whatever conclusion was intended.The former Supplementary Figure 4E supports the point that not all L4-specific and L5-specific genes are affected by the knockout, which one would expect if the change we observe were simply due to L5 contamination (a concern raised by reviewers in the first round of comments). This panel is now moved to Main Figure 4E. We have added a statistical analysis (Fisher exact test) to show that although only a subset of L4 and L5 genes are affected, downregulation of L4 genes and upregulation of L5 genes are highly overrepresented . As explained in the legend and the text, these include all of the genes with 1.5 L4:L5 fold-change and expression > 1.6 as identified by the Allen Brain Atlas differential search. Since these genes exclude the 31 L4 genes and 50 L5 differentially expressed genes shown in Figure 4A-C, they are by definition, not statistically altered by the knockout, as shown by the similarity of the mean fold-change (black line) to the gray line indicating no change. Therefore, this new analysis aims to make two points: (1) the data are not consistent with simple contamination of Rorb KO samples by L5 cells, and (2) provide additional statistical support for the conclusion that L4 and L5 genes are significantly altered. We have added clarification of these results to the legend and to the Results subsection “RORβ is required for expression of a layer 4 gene profile and repression of layer 5 genes”.


DNA viruses can hijack and manipulate the host chromatin state to facilitate their infection. Multiple lines of evidences reveal that DNA virus infection results in the host chromatin relocation, yet there is little known about the effects of viral infection on the architecture of host chromatin. Here, a combination of epigenomic, transcriptomic and biochemical assays were conducted to investigate the temporal dynamics of chromatin accessibility in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The high-quality ATAC-seq data indicated that progressive chromatin remodeling took place following BmNPV infection. Viral infection resulted in a more open chromatin architecture, along with the marginalization of host genome and nucleosome disassembly. Moreover, our results revealed that chromatin accessibility in uninfected cells was regulated by euchromatic modifications, whereas the viral-induced highly accessible chromatin regions were originally associated with facultative heterochromatic modification. Overall, our findings illustrate for the first time the organization and accessibility of host chromatin in BmNPV-infected cells, which lay the foundation for future studies on epigenomic regulation mediated by DNA viruses. As a well-studied arthropod-specific double-stranded DNA virus, Bombyx mori nucleopolyhedrovirus (BmNPV) is a representative member of baculoviruses. BmNPV infection results in significant host chromatin marginalization, which has also been found in most DNA viruses. However, the effects of baculovirus infection on the organization and accessibility of host chromatin are poorly understood. Here, by using ATAC-seq, we show that DNA virus BmNPV infection gradually remodels the accessibility of host chromatin. ATAC-seq data reveal that the marginalized host chromatin is a more accessible architecture along with the depletion of multi-nucleosome depositions. Moreover, our findings suggest the increased accessibility regions are regulated by the facultative heterochromatic modification. Overall, we provide a novel understanding of molecular mechanisms by which baculovirus and DNA viruses alter the organization of host chromatin in epigenomic regulation. DNA viruses are nucleic acid-based obligate intracellular microorganisms, which commonly replicate and assemble in the host nucleus. In the process of infection, the viral genome amplifies in an independent replication compartment and the host genome is marginalized to the nuclear periphery –6. A numBombyx mori, which can cause severe damage to the sericulture industry around the world [Baculoviruses are a large group of invertebrate-specific DNA viruses with rod-shaped nucleocapsids, which play vital roles in insect pest control and heterologous protein expression . Bombyx he world . BmNPV phe world . SimilarIn the course of baculovirus infection, viral DNA is synthesized in the virogenic stroma (VS), and the host chromatin is marginalized as a result of VS expansion , 11. VS Although baculovirus infection caused host genome marginalization has been characterized, the states of chromatin accessibility and organization remain largely unknown, even though they have been mentioned in a previous study . Little In order to better understand how baculovirus manipulates the host genome, in this report we describe the systematic exploration of the dynamic changes in silkworm chromatin during BmNPV infection by using a combination of epigenetic, transcriptomic, and biochemical assays. According to our results, the host chromatin was remarkably marginalized at the very late stage of infection. Furthermore, ATAC-seq was employed to characterize genome organization, especially for the DNA accessibility of control as well as BmNPV-infected cells at designated time points . As the viral infection progressed, the numbers of differentially accessible peaks increased, and there were more accessible regions in the marginalized host chromatin, with disappeared multi-nucleosome deposition. Finally, we compared the public ChIP-seq data with our ATAC-seq data, which indicated that histone epigenetic modifications were involved in the organization of host genome. Taken together, these results not only illustrate the global profiling of host chromatin accessibility and architecture during BmNPV infection, but also shed new light on the regulation of host epigenomic dynamics in response to baculovirus infection. Meanwhile, our new genome-scale observations directly reveal the organization of marginalized chromatin, which will greatly advance the research of DNA viruses in this aspect.Fig 1A), suggesting that histone H3 was integrated with host chromatin and can be used as an effective marker of chromosome distribution. Following VS expansion from 8 to 48 h p.i., the host genome was gradually located at the lamin-labeled inside region of nuclear membrane . To further characterize the alteration of nuclear ultrastructure, we used the high-pressure freeze (HPF) and freeze substitution (FS) techniques in combination with transmission electron microscopy (TEM) [Fig 1B, VS occupied the nuclear center, and numerous electron-dense depositions were distributed in the nucleoplasm around VS at 24 h p.i. By 48 h p.i., VS had gradually expanded concurrent with the marginalization of electron-dense depositions around the perinuclear membrane , indicating that these depositions were host chromatin. The central region of the nucleus was left for nucleocapsid assembly and ODV envelopment. Taken together, these results demonstrated that BmNPV infection resulted in host chromatin extruded into the inside region of nuclear membrane, which was thought to change the host genome organization.It is well known that the baculovirus-induced VS is the region responsible for both viral DNA replication and nucleocapsid assembly . Althougpy (TEM)  to obserDrosophila melanogaster embryonic ATAC-seq profiles [S1A Fig), which was different from the results in mammals [S1B Fig). The accessible regions were visualized and quantified, which suggested that the obtained data were of high-quality with a strong and stable signal density to background .In eukaryote cells, genome is dynamic and non-randomly organized in the nucleus. Therefore, it was surmised in this study that DNA accessibility was dynamically remodeled during host chromatin marginalization. To confirm our hypothesis, we applied ATAC-seq technology to investigate the effects of BmNPV infection on the global host chromatin accessibility in BmN cells. According to our ATAC-seq data, consistent with data from the profiles , BmN cel mammals . BesidesFig 2A). In addition, principal components analysis (PCA) also exhibited a significantly positive similarity among the open chromatin landscapes in each group and progressive changes in BmNPV-infected cells, which suggested that viral infection resulted in substantial chromatin remodeling . To further characterize the genomic functional elements of accessible regions, HOMER [Fig 2C) during infection. In contrast, the enrichment of promoter-TSS gradually decreased from 18 to 8% as the viral infection progressed . Additionally, the genome-wide chromatin accessibility was visualized through comparing those regions of the top 30 size scaffolds among all samples . This circle diagram clearly displayed the radical and global alterations of chromatin accessibility in these samples in response to BmNPV infection . To sum up, these ATAC-seq data demonstrated that BmNPV infection induced dynamic changes in the chromatin accessibility.Statistical analysis revealed that the number of accessible peaks was higher in BmNPV-infected cells than in control group, which was especially significant at 48 h p.i. . Notably, over 23,000 ATAC-seq peaks gained accessibility at 48 h p.i., and they were specifically enriched in intergenic and intron regions, but less enriched in promoter-TSS and exon elements. In contrast, peaks that were more accessible in 8 and 24 h p.i. groups were preferentially enriched in promoter-TSS and exon elements , suggesting that these elements might have greater effects on regulating gene expression at early and late infection phases. Among peaks that were accessible in any of these groups, few (n = 642) were differentially accessible between the control and 8 h p.i. group ; however, such figure was dramatically changed to about 3,000 DAPs between the control and 24 h p.i. group , and over 25,000 DAPs were identified between the control and 48 h p.i. group . As shown in Fig 3C, we found that accessibility of all peak has no significant correlation with expression of genes. In addition, the relationships between gene expression and chromatin accessibility were weak across genes that gained or lost accessibility in promoter-TSS elements at 8 and 24 h p.i. , indicating that the accessibility at promoter-TSS was not a major element responsible for transcriptional regulation in response to viral infection at the early or late stages. However, when the infection progressed to 48 h p.i., gene expression was highly regulated by chromatin accessibility at promoter-TSS elements .To identify the specific dynamic changes in the chromatin accessibility landscape in response to viral infection, limma  was usedS3A–S3F Fig). Typically, GO term related to innate immune response was enriched in the opening sites at 8 h p.i. , which showed that the innate immune pathways were activated at the early stage to defend against viral infection. For instance, the promoter-TSS and upstream distal elements in the LOC101735468, a toll-like receptor 5 homologue in Bombyx mori, gained accessibility at 8 h p.i. . These findings were consistent with previous studies reporting that baculovirus infection activated the host Toll innate immune pathway in both mammals and invertebrates [Fig 4C and S3D and S3F Fig). By comparing the RNA-seq data, and the gene loci that lost density, we observed a statistically significant decline in gene expression during viral infection . In addition, the cell detachment assay [Fig 3D). Further, the loci of SCB (LOC101746113), an integrin alpha PS3 (Drosophila melanogaster) homologue, exhibited a deceasing trend in both DNA accessibility and gene expression . The above results collectively revealed that BmNPV infection epigenetically suppressed the expression of cell adhesion genes and induced host cell detachment.To further identify the roles of these DAPs in viral infection, the functions of DAPs-corresponding genes were annotated based on gene ontology (GO) analysis . After assessing the genomic features of these cluster peaks, only 4.2% peaks were found to be distributed in promoter-TSS elements of cluster II, which was substantially less than other clusters .In contrast to a previous study reported that no alteration in the host chromatin structure , our finS4A and S4C Fig). These enriched GO terms were also observed at sites that gained accessibility at 24 and 48 h p.i. groups , which indicated that BmNPV infection activated the host transcription and translation systems to facilitate viral gene expression. For example, the regulatory elements associated with the promoter-TSS region of ribosomal protein L21 (RpL21), a component of the large ribosomal subunit, were gradually up-regulated in cluster I . Another example from cluster IV includes the upstream distal element of gene E2F transcription factor 1, E2F1, was up-regulated at 48 h p.i. . The protein encoded by E2F1 is a transcription factor involved in several host responses induced by baculovirus infection, such as, regulation of apoptosis [Cluster I was comprised of 5,936 peaks that gradually gained accessibility as a response to viral infection; and cluster IV contained 12,333 peaks with increasing accessibility by 48 h p.i. These regions might reflect the host chromatin response activated by BmNPV infection. GO analysis showed that the peaks in cluster I were highly enriched in translation and transcription related genes, such as those involved in translation, post-translational protein modification, and transcription (DNA-templated); in addition, regulation of transcription (DNA-templated) was enriched in cluster IV . This observation suggested that viral infection led to numerous regions that uniquely gained accessibility at 48 h p.i., and such result was similar to the differential analysis showing that more than 23,000 peaks gained accessibility at 48 h p.i. . Although fewer promoter-TSS elements were enriched in this cluster, the genes close to these elements were highly expressed in 48 h p.i. group as evidenced by comparing the RNA-seq data of two representative GO terms . Consequently, the above results indicated that peaks in cluster II broadly and uniquely gained accessibility at 48 h p.i.Cluster II was comprised of more than 36,000 peaks that were specifically accessible in 48 h p.i. group, but had a very low signal in both the control, 8 and 24 h p.i. groups . GO analysis showed that elements in this cluster were highly enriched for genes associated with nucleosome assembly, which was also observed in GO functions that lost accessibility at 24 and 48 h p.i. . Nonetheless, the expression of nucleosome assembly-associated genes was not significantly down-regulated, as evidence by the RNA-seq data . Surprisingly, the GO term associated with chromatin assembly or disassembly was enriched in regions that gained accessibility at 48 h p.i. . Transcriptomic analysis revealed that the expression of these genes was remarkably up-regulated in 48 h p.i. group . Prime examples of this group including elements in CBX5 (LOC101740470) and ASF1 (LOC101742242) loci, encoding chromobox protein homolog 5 (CBX5) and histone chaperone asf1(ASF1) respectively, became strongly accessible at 48 h p.i. . qRT-PCR results confirmed that these two genes were significantly up-regulated at 48 h p.i. . CBX5 is a heterochromatin component that drives heterochromatin distribution to the inner nuclear membrane through interacting with lamin-B receptor (LBR) [Cluster III consisted of 6,985 regions that gradually lost accessibility during viral infection (or (LBR) , 33. Moror (LBR) , 35.Taken together, these results indicated that BmNPV infection induced certain genome regions to gain accessibility and these regions positively regulated gene expression at 48 h p.i.in vivo DNase I TUNEL assay [Fig 5A). These results verified our conclusion that chromatin accessibility broadly increased following BmNPV challenge.To further investigate the genome-wide chromatin organization remodeling, we carried out the EL assay  to detecFig 5B). To identify the detailed changes in nucleosome organization, we used the insert size density plot (V-plot) to calculate the visualization of ATAC-seq data. The position of flanking nucleosomes in the control showed an enrichment of short fragments (less than 140 bp) in clusters I, III and IV, together with an enrichment of mono-nucleosomal size fragments (150–200 bp) in cluster II. These results indicated that the chromatin in cluster II was compactly organized by nucleosomes and less accessible, however, the regions in other clusters were accessible in the control . For virus-infected cells at 48 h p.i., the insert size mostly concentrated in the short fragments across all clusters . In cluster II, the short fragments were only enriched at 48 h p.i. , demonstrating that the regions in this cluster lost nucleosome deposition and gained accessibility. Moreover, the expression of histone H3 was down-regulated at 48 h p.i. , which was also observed in BmNPV-infected Bombyx mori pupae [Chromatin organization is a fundamental biological process mediated by nucleosome assembly and disassembly in eukaryotic genomes , 38. Theri pupae . The aboS5A–S5C Fig). Moreover, the H3K4me3 and H3K27ac profiles displayed a similar landscape pattern to that of the open chromatin regions, particularly enriched in clusters I, III and IV and lost signal in cluster II . In contrast, H3K27me3 had signals in cluster II regions . Thus, the concentrations of DNA-associated H3K27ac and H3K4me3 were dramatically higher than those of H3K27me3 in clusters I, III and IV sites, whereas that of H3K27me3 was higher in cluster II regions than those of the other two modifications . These results suggested that the open chromatin regions significantly overlapped with H3K4me3 and H3K27ac, indicating that these two euchromatin markers were involved in the accessible chromatin organization. As a heterochromatin marker, however, H3K27me3 participated in the maintenance of epigenetic repression state in cluster II regions in the control, and this cluster was activated at the very late stage of BmNPV infection.Previous studies showed that epigenetic modifications of histone H3 in the silkworm were similar to other eukaryotes, and H3K4me3, H3K27ac were most prominent euchromatin markers, whereas H3K9me3 and H3K27me3 represented heterochromatin regions in BmN cell line –42. In eS5D Fig, H3K27ac was up-regulated and the other methylated histone forms were down-regulated at 48 h p.i., indicating that H3K27ac might be involved in the BmNPV infection-induced widespread gain accessibility. Thus, ChIP-seq was conducted to test the relationship between H3K27ac and DNA accessibility during virus infection. As expected, H3K27ac in clusters I and II regions had higher signals in 48 h p.i. group than in control group . In contrast, the enrichments of H3K27ac in clusters III and IV sites were markedly higher in BmNPV infected cells than in uninfected cells . These results suggested that H3K27ac exerted essential roles in viral-induced the regions of clusters I and II that gained accessibility at the very late stage of infection.Subsequently, to determine the functions of histone modifications in regulating chromatin accessibility upon BmNPV infection, we detected the levels of these modifications. As shown in Fig 8). These results contribute to explaining the effects of viral infection on the dynamic changes in chromatin accessibility and organization, and lay the important basis to explore the virus-host interactions, especially the epigenome mechanism during the viral life cycle.In this study, we performed ATAC-seq approach to investigate the BmNPV infection mediated changes in genome organization and accessibility. Epigenome, transcriptome and biochemical assays were carried out to comprehensively examine the host chromatin accessible landscape in response to baculovirus infection. As revealed by our data, radical genome remodeling and a broad increase in chromatin accessibility occurred concomitantly with nucleosome disassembly when the host genome was marginalized during viral infection . In addition, LBR is a part of the nuclear lamina (NL) and NL closely contacts with specific genomic regions, which is termed as lamina-associated domains (LADs) [Many DNA viruses replicate in the intranuclear viral replication compartment (VRC), and viral DNA can directly/indirectly interact with the host genome –45. The itecture . CBX5 coitecture , as a reitecture , 33. Thes (LADs) , 48. NL s (LADs) . In bacus (LADs) . Thus, iASF1 locus encodes a histone chaperone that facilitates histone deposition. Our results confirmed the speculation that the increased accessibility and up-regulated expression at ASF1 locus participated in the chromatin disassembly at the late infection . The alterations of DNA accessibility and nucleosome assembly are suggested to affect the 3D genome structure [The increased peak numbers and DAPs illustrated that substantial chromatin remodeling occurred in the BmNPV-infected cells. A recent study in yeast reveals that positioning heterochromatin at the nuclear periphery facilitates epigenetic suppression . Howevertructure , includitructure –55. The tructure  and ChIAtructure , shed moS2L Fig). The pathogenicity of viruses normally causes cytotoxicity and tissue disruption, and viral envelope proteins have exerted essential functions in virulence and cell detachment [In addition, functional annotations of promoter-TSS regions combined with transcriptome analysis are critical to explore the distinct epigenetic signatures between BmN cells and BmNPV-infected cells. For instance, our results indicated that genes within the cell adhesion term lost accessibility and decreased expression during viral infection, suggesting that the host cells had a reduced ability to attach to the substrate. Indeed, our findings were supported by the cell detachment assay . The results indicated that, H3K4me3 and H3K27me3 were markedly down-regulated at the very late stage of BmNPV infection, but H3K27ac was up-regulated, suggesting that the increase of H3K27ac as well as the decrease of H3K27me3 might contribute to the elevated DNA accessibility. Similar results have been reported for the Epstein-Barr virus (EBV) infected human primary B cells [To extend our insight into the changes of DNA accessibility in epigenetics, the relationships among the H3K4me3, H3K27ac, and H3K27me3 profiles were analyzed based on our ATAC-seq data. The public ChIP-seq data indicated that euchromatic markers (H3K4me3 and H3K27ac) were strongly correlated with the open chromatin regions, whereas heterochromatic modification (H3K27me3) showed less overlap, demonstrating that H3K4me3 and H3K27ac were necessary for maintaining chromatin accessibility in insect,  B cells . EBV infTo the best of our knowledge, this study has first reported the dynamic profiles of chromatin accessibility and architecture in response to baculovirus infection, which will help to provide a deeper insight into the virus-induced epigenomic regulation, and uncover this conserved mechanism from vertebrate to invertebrate DNA viruses.6 BmN cells were inoculated with WT-BmNPV  for 1 h at 27°C, then, the supernatant was replaced with fresh Sf-900 II SFM medium, and this time point was defined as 0 h p.i. Afterwards, cells were harvested at indicated time points  for subsequent ATAC-seq, RNA-seq and biochemical assays.BmN cells were maintained as a monolayer in the Sf-900 II SFM medium (Invitrogen Life Technologies) supplemented with 3% fetal bovine serum . The T3 strain of BmNPV was used as wild type (WT) virus in this study. Briefly, 5×10Briefly, cells on the coverslips were infected with WT virus at an MOI of 10. Thereafter, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% TritonX-100 for 20 min, blocked in the PBS solution containing 5% BSA for 1 h, and incubated with primary rabbit polyclonal anti-Histone H3 antibody (Abcam) and mouse monoclonal anti-Lamin Dm0 antibody (DHSB) overnight at 4°C at indicated time points. Afterwards, cells were incubated with secondary Alexa 488-conjugated goat anti-rabbit antibody and Alexa 546-conjugated goat anti-mouse antibody (Invitrogen). Finally, cells were incubated with 4’,6-diamidino-2-phenylindole , and examined through ZEISS LSM 780 confocal scanning laser microscopy (CSLM).2 (Leica). Afterwards, the vitrified cells were preserved in liquid nitrogen and later placed in the FS cocktail flasks at -90°C in the Automate Freeze-Substitution (AFS). The substitution medium was comprised of 2% OsO4 and 2.5% H2O in acetone, and FS was carried out in accordance with the following procedure: -90°C for 24 h; heating to -60°C at a rate of 2°C/h; -60°C for 8 h; heating to -30°C at a rate of 2°C/h; -30°C for 8 h; and heating in pure acetone for thrice at an interval of 10 min. Finally, the samples were progressively embedded within the epoxy-acetone mixture (1:3) for 2 h at -30°C before heating to 0°C at a rate of 15°C/h; then within the epoxy-acetone mixture (1:1) for 2 h at room temperature; and eventually within the epoxy/acetone mixture (3:1) overnight at room temperature. Later, samples were incubated in pure epoxy for 2 h thrice, and finally in a new bath of pure epoxy overnight prior to polymerization at 60°C for 24 h. Thereafter, the 70-nm ultrathin sections were cut using a diamond knife (45° angle) on the Leica UC6 ultramicrotome, and collected onto the carbon-coated grids. Eventually, the samples were observed at room temperature using the TEM Tecnai G2 spirit at 120 kV.High-pressure frozen (HPF) and freeze substitution (FS) was performed in combination with TEM, as previously reported . In brie4 cells were lysed in 50 μl cold lysis buffer  to obtain the nuclei for each sample. Later, the Nextera DNA Sample Preparation Kit (Illumina) was adopted for carrying out the Tn-5 transposition reaction at 37°C for 30 min. Then, DNA fragments were purified through the MinElute Kit (Qiagen), and amplified for 5 cycles using those previously designed primers containing both compatible adaptors and barcodes. Subsequently, the resulting ATAC-seq libraries were purified , and the final libraries were sequenced on the NovaSeq 6000 illumina platform with a paired-end read of 150 bp.ATAC-seq libraries were prepared as described previously . In brieChIP-seq libraries were prepared as described previously . Cells wAfterwards, the total RNA was isolated from the harvested cells using RNAiso Plus (Takara) according to the manufacturer instructions. Three microgram of RNA per sample was used as the input material for library preparation, the RNA-seq libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB), and index codes were added to attribute the sequences to each sample. Then, the as-obtained RNA-seq libraries were sequenced on the HiSeq X ten illumina platform with a paired-end read of 150 bp.Bombyx mori genome and BmNPV genome using BWA [ATAC-seq data were processed using the Kundaje Lab’s ATAC-seq pipeline  . The significant GO categories were identified through Fisher’s exact test, and FDR was adopted to correct the p-values.For functional analysis, GO annotations were downloaded from agriGO  = (floating cells/ (floating cells + adherent cells) × 100%).For immunoblotting assay, 30 μg cellular proteins at indicated time points were mixed with 2× Laemmli buffer, separated through 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and examined through western blotting using the rabbit polyclonal anti-Histone H3 antibody (Abcam), rabbit polyclonal anti-α-tubulin antibody (Beyotime), mouse monoclonal anti-GP64 antibody (Sigma), rabbit polyclonal anti-H3K4me3 antibody (Abcam), rabbit polyclonal anti-H3K27ac antibody (Abcam), and rabbit polyclonal anti-H3K27me3 antibody (Active motif).Tua2-F: 5’-TGCCCGAGGACACTATACCA-3’, Tua2-R: 5’- ACACGAGGAAACCCTGAAGC-3’, asf1-F: 5’-CGCCAATGGAATGCTGAGATTAT-3’, asf1-R: 5’-GTACAAACAAAGCCTTGAAAACGG-3’, cbx5-F: 5’-ATCCATTGGAAGGGCTGGTC-3’, cbx5-R: 5’-GGCGCTGCTCACTTTATCCA-3’.For qRT-PCR analysis, BmN cells and BmNPV-infected cells at indicated time were harvested and total RNA was extracted by using TRIzol reagent (Invitrogen). 5μg total RNA was used to synthesis the first-strand cDNAs by TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen). qRT-PCR was performed using Hieff qPCR SYBR Green Master Mix (Yeasen) and the primers of qRT-PCR were used as follows: In vivo DNase I & TUNEL assays were carried out as described previously  with milPearson’s correlation analysis was performed with the R platform. Student’s t-test and two-way ANOVA were conducted using the GraphPad Prism6. P<0.05 indicated statistical significance.https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJNA549316 and PRJNA628845. The histone modifications ChIP-seq data were obtained from the NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra) under accession number DRX001335, DRX016832 and DRX016833.The ATAC-seq, RNA-seq and ChIP-seq data generated in this study have been submitted to the NCBI BioProject database  The fragment length histograms of the two replicates of BmN cells indicate the expected pattern of nucleosomal transposase insert sizes. (B) Scatter plots showing the ATAC-seq reads correlation coefficient between replicates of each group. (C) IGV genome browser view of eIF 2a kinase gene ATAC-seq signal profiles.(TIF)Click here for additional data file.S2 Fig(A) Distribution of genomic features of opened or closed peaks during BmNPV infection. (B) Volcano plot of differentially accessible peaks between BmN cells with 8 and 24 h p.i. group. (C) Cumulative distribution function of peak accessibility changes between BmN cells and the 8 h p.i. group. (D) Cumulative distribution function of peak accessibility changes between BmN cells and 24 h p.i. group. (E) Cumulative distribution function of peak accessibility changes between BmN cells and 48 h p.i. group. (F) Normalized ATAC-seq profiles at LOC101735468 loci. Shaded regions are representative of an increase at 8 h p.i.(TIF)Click here for additional data file.S3 Fig(A-F) Biological processes (GO terms) enriched in genes which correspond to gained at 8 h p.i., lost at 8 h p.i., gained at 24 h p.i., lost at 24 h p.i., gained at 48 h p.i., and lost at 48 h p.i., respectively.(TIF)Click here for additional data file.S4 Fig(A-C) Biological processes (GO terms) enriched in genes which correspond to cluster I, cluster II and cluster IV, respectively.  Normalized ATAC-seq profiles at RpL21and E2f1 loci, respectively. Shaded regions represent increase during BmNPV infection.  Boxplots of G-protein coupled receptor signaling pathway and proteolysis associated gene expression in all groups. Two-way ANOVA was performed between BmN and 48 h p.i. groups. (H) The heatmap shows the expression of genes related to nucleosome assembly GO term during BmNPV infection.(TIF)Click here for additional data file.S5 Fig(A-C) Venn diagrams showing the overlaps between histone modifications and ATAC-seq peaks in BmN cells. (D) BmN cells and BmNPV-infected cells were analyzed by western blot with the indicated histone modifications antibodies.(TIF)Click here for additional data file.


Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulatory factor of preadipocyte differentiation. As a result of alternative splicing and alternative promoter usage, PPARγ gene generates multiple transcript variants encoding two protein isoforms. Krüppel-like factor 2 (KLF2) plays a negative role in preadipocyte differentiation. However, its underlying mechanism remains incompletely understood. Here, we demonstrated that KLF2 inhibited the P1 promoter activity of the chicken PPARγ gene. Bioinformatics analysis showed that the P1 promoter harbored a conserved putative KLF2 binding site, and mutation analysis showed that the KLF2 binding site was required for the KLF2-mediated transcription inhibition of the P1 promoter. ChIP, EMSA, and reporter gene assays showed that KLF2 could directly bind to the P1 promoter regardless of methylation status and reduced the P1 promoter activity. Consistently, histone modification analysis showed that H3K9me2 was enriched and H3K27ac was depleted in the P1 promoter upon KLF2 overexpression in ICP1 cells. Furthermore, gene expression analysis showed that KLF2 overexpression reduced the endogenous expression of PPARγ transcript variant 1 (PPARγ1), which is driven by the P1 promoter, in DF1 and ICP1 cells, and that the inhibition of ICP1 cell differentiation by KLF2 overexpression was accompanied by the downregulation of PPARγ1 expression. Taken together, our results demonstrated that KLF2 inhibits chicken preadipocyte differentiation at least inpart via direct downregulation of PPARγ1 expression. Adipose tissue is not only a depot for energy storage but is also the body's largest endocrine organ and regulates multiple physiological processes by secreting various endocrine and paracrine factors Ràfols, . AdiposeIn vivo and in vitro studies have shown that many of the Sp1/KLF family members play critical roles in preadipocyte differentiation  and two protein isoforms (PPARγ1 and PPARγ2) . The P1 promoter fragment (−327 to +108 bp) was obtained by double digestion of the amplified pGL3P1-327/+108 with KpnI and HindIII and was mock-treated or treated with M.SssI methylases  to yield the unmethylated and methylated P1 promoter fragments (-327 to +108 bp) according to the manufacturer's instructions. Methylation was confirmed by restriction enzyme digestion using BstUI , which recognizes CGCG and is sensitive to methylation. The resulting unmethylated and methylated P1 promoter fragments (−327 to +108 bp) were religated into the pGL3-Basic vector to generate the unmethylated and methylated pGL3P1-327/+108.The KLF2 expression plasmid (pCMV-Myc-KLF2) was previously constructed by our lab . The luciferase activities were determined at 48 h after cotransfection by a Dual-Luciferase Reporter System .The DF1 or ICP1 cells were washed with cold PBS and lysed using RIPA Buffer containing 1% PMSF . Cellular proteins were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes . Then the membranes were blocked for 60 min and incubated overnight at 4°C with a Myc-specific antibody , a PPARγ antibody  or a β-actin antibody  as the primary antibody. After the blots were rinsed with PBST three times, they were incubated with HRP-conjugated anti-mouse  for 60 min at room temperature as the secondary antibody. The blots were observed with an ECL Plus detection kit .ChIP was conducted using a ChIP assay kit  as described previously  and the H3K27ac antibody , and mouse IgG  and rabbit IgG  were used as controls for the H3K9me2 and H3K27ac antibodies, respectively. The rest of the ChIP procedure was the same as described above.CGGTGCCTGGCCGGTAGGATGGGCCGGCCCG, where the four CpG sites, which are underlined, were unmethylated), while the biotin-labeled methylated oligonucleotide probes were named the CpG met4 probe  and the CpG met1 probe . The sequences of the sense and antisense probes are shown in The KLF2 nuclear proteins were isolated from DF1 cells transfected with the pCMV-Myc-KLF2 construct using the nuclear extraction kit . The binding capacity of KLF2 to the DNA was detected by an EMSA Kit . Unmethylated and methylated biotin-labeled probes harboring the potential KLF2 binding site and the corresponding cold and mutant probes containing the P1 promoter sequence were synthesized by Genewiz . The biotin-labeled unmethylated oligonucleotide probe was named the CpG probe . RT-qPCR was performed using a SYBR Green PCR Master Mix  on the 7500 Real-Time PCR System . The NONO gene was used as an internal control. The expression data were calculated using the 2CT−ΔΔ relative quantification method. The primers used are shown in The total RNA was isolated from DF1 or ICP1 cells using TRIzol reagent , and the cDNA was generated with the ImProm-IIt-tests by Graph Pad Prism 5. The p values < 0.05 were considered significant .Data are expressed as the mean ± standard error of the mean (SEM). Comparisons between groups were performed using unpaired two-tailed Student's p < 0.05 or p < 0.01)  or its series of 5′ deletion mutant constructs with the pCMV-Myc-KLF2 or empty pCMV-Myc vector plus the pRL-TK into DF1 or ICP1 cells. The reporter gene assay showed that compared to transfection of the empty vector pCMV-Myc, transfection with pCMV-Myc-KLF2 significantly repressed the luciferase reporter activity of the P1 promoter (pGL3P1-1891/+108) and its five 5' deletion mutants in both DF1 and ICP1 cells ( < 0.01) . These dp < 0.01), but transfection of pCMV-Myc-KLF2 had no inhibitory effect on the promoter activity of pGL3P1+1/+108MUT  was present at positions +18 to +28 bp. To demonstrate that the putative Sp1/KLF binding site is required for the KLF2-mediated transcription inhibition of the P1 promoter, we replaced this putative Sp1/KLF binding site with GAGTATTTGAA in the P1 promoter construct (pGL3P1+1/+108) , and the/+108MUT . Togethep < 0.01) (p > 0.05). Collectively, these results suggest that KLF2 can directly bind to and regulate the P1 promoter that covered the P1 promoter region (-60 to +52 bp) . The res < 0.01) . In contpromoter .p < 0.05, data not shown). However, further pyrosequencing analysis showed that the methylation level of the CpG site located in the KLF2 binding site . There are only 9 nucleotides between CpG1 and CpG2, whose methylation level was significantly higher in the fat chicken lines than in the lean chicken lines. These findings prompted us to test whether DNA methylation affects the KLF2-mediated regulation of the P1 promoter. To determine whether DNA methylation influences the KLF2-mediated suppression of P1 promoter activity, we performed a reporter assay. The unmethylated and methylated P1 promoter reporters (pGL3P1-321/+108) were individually transfected into DF1 cells with pCMV-Myc or pCMV-Myc-KLF2 plus pRL-TK. Notably, the results showed that KLF2 could efficiently inhibit the activities of both the unmethylated and methylated P1 promoters (p < 0.01) (p > 0.05).Our bioinformatics analysis revealed that the P1 promoter contained a CpG island at−333 to +52 bp and that the identified KLF2 binding site was located within this CpG island. This KLF2 binding site (+18 to +28 bp) contained a CpG site (underlined)  designatte CpG1,  was not  < 0.01) . KLF2 teThen, we further determined whether DNA methylation affects the binding of KLF2 to the P1 promoter by EMSA. The labeled P1 probe (+21 to +54 bp) (here after referred to as CpG probe), including CpGs 1–4, and two methylated counterpart probes  were used in EMSAs. The EMSA results showed that a specific shifted band was observed with any of the three probes  , lane 1.Becn1 in RAW264.7 cells, a mouse monocytic cell line, resulting in up- or downregulation of Becn1 expression  on the promoter of PPARγ1, which is driven by the P1 promoter, in DF1 and ICP1 cells. pCMV-Myc-KLF2 or pCMV-Myc was transfected into DF1 and ICP1 cells, and after 48 h of transfection, the endogenous expression of PPARγ1 and the PPARγ protein level were determined using qRT-PCR and western blots. As expected, KLF2 protein was expressed in the pCMV-Myc-KLF2-transfected DF1 and ICP1 cells but not in the empty pCMV-Myc vector-transfected cells at 48 h after transfection. The qRT-PCR analysis showed that the endogenous PPARγ1 expression was decreased by 27.22 and 26.83%, respectively, in DF1 and ICP1 cells transfected with pCMV-Myc-KLF2, compared with the cells transfected with the empty pCMV-Myc , FABP4 (72 h), and AdipoQ (72 h)  , but KLFPPARγ transcriptional regulation, it is worth investigating whether these transcription factors regulate the P1 promoter through competition in the future.Several transcription factor binding sites overlap or near the vicinity of the KLF2 binding site on the P1 promoter   of chicken PPARγ gene simultaneously or sequentially.Our previous study showed chicken PPARγ2 promoter and inhibited the 3T3-L1 differentiation (Banerjee et al., PPARγ2 promoter activity (Banerjee et al., Our present study showed that KLF2 repressed the P1 promoter activity and inhibited chicken preadipocyte differentiation. Similar to our findings, Banerjee et al. demonstrated that KLF2 remarkably reduced the activity of mouse Promoter DNA methylation is negatively or positively correlated with gene expression (Adam and Farnham, BECN1 (Laha et al., Transcription factors result in histone modifications in their target promoters by recruiting histone methylases/demethylases or histone acetylases/deacetylases (Liu et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.TC performed the experiments, analyzed the data, and wrote the manuscript. NW conceived and supervised the study. NW and TC designed the experiments. JH and BN participated in the real-time PCR analysis. FM participated in western blot analysis. YS participated in the data analysis. All authors revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.