The blood-brain barrier (BBB) controls the movement of molecules into and out of the central nervous system (CNS). Since a functional BBB forms by mouse embryonic day E15.5, we reasoned that gene cohorts expressed in CNS endothelial cells (EC) at E13.5 contribute to BBB formation, whereas adult gene signatures reflect BBB maintenance mechanisms. Supporting this hypothesis, transcriptomic analysis revealed distinct cohorts of EC genes during BBB formation and maintenance. Here we demonstrate that epigenetic regulator’s histone deacetylase 2 (HDAC2) and polycomb repressive complex 2 (PRC2) control EC gene expression for BBB development and prevented Wnt/β-catenin (Wnt) target genes from being expressed in adult CNS ECs. Low Wnt activity during development modifies BBB genes epigenetically for the formation of functional BBB. As a Class-I HDAC inhibitor induces adult CNS ECs to regain Wnt activity and BBB genetic signatures that support BBB formation, our results inform strategies to promote BBB repair.
The specific questions taken up for study by the authors-in mice of HDAC and Polycomb function in the context of vascular endothelial cell (EC) gene expression relevant to the blood-brain barrier, (BBB)-are potentially useful in the context of vascular diversification in understanding and remedying situations where BBB function is compromised. The strength of the evidence presented is incomplete, and to elaborate, it is known that the culturing of endothelial cells can have a strong effect on gene expression. This is a fundamental issue as we are not given how long the cells were cultured and how the above point was addressed.
Central nervous system vessels possess a blood-brain barrier (BBB) that prevents toxins and pathogens from entering the brain. A leaky BBB can lead to deleterious consequences for CNS disorders including stroke, traumatic brain injury, and brain tumors1. Currently, no treatment options are available to sustain and/or regenerate BBB integrity. The identification and targeting of the mechanism that forms and maintains the BBB is an attractive strategy to regain BBB integrity.
By comparing the EC transcriptome of peripheral and brain vessels, the genomic profile that contributes to BBB was described2,3. Further, extensive gene expression changes in CNS ECs during development were also reported 4,5. However, molecular mechanisms governing BBB gene transcription and how such mechanisms contribute to BBB establishment and maintenance are unresolved.
Epigenetic modifications of histones and chromatin-modifying enzyme activities are critical determinants of gene expression6. Histone deacetylases (HDACs) associate with specific transcription factors and participate in gene repression7. HDAC inhibitors are important experimental tools for elucidating HDAC functions8, and four HDAC inhibitors are FDA-approved drugs for cancer treatment9. More than thirty HDAC inhibitors are being investigated in clinical trials10. Similarly, polycomb repressive complex 2 (PRC2), a complex of polycomb-group proteins (such as EZH2, EED, and SUZ12), represses transcription via its methyltransferase activity that catalyzes tri-methylation of H3K2711,12. An EZH2 inhibitor is FDA-approved13 for Follicular lymphoma.
Wnt/β-catenin (Wnt) signaling is essential for establishing the BBB 4,5,14-16, but the activity of this pathway declines gradually thereafter and is reported to be minimal in adults15,17,18. Evidence linking Wnt to the regulation of BBB genes includes an EC-specific knockout (KO) of β-catenin that affects BBB gene expression and integrity5,19-21. In CNS ECs, Wnt signaling requires the binding of Wnt ligands Wnt7a/7b to Frizzled receptors. This interaction stabilizes the intracellular signaling molecule β-catenin, by suppressing a cytoplasmic destruction complex, which would otherwise degrade β-catenin. Stabilized β-catenin translocates to the nucleus and regulates Wnt target gene transcription by interacting with DNA binding transcription factors TCF/LEF (T-cell factor/lymphoid enhancing factor). The mechanisms underlying the reduced Wnt signaling and how Wnt regulates the BBB gene transcription are not established.
We describe the discovery of epigenetic mechanisms responsible for regulating BBB gene expression during development, how Wnt signaling is reduced, and the significance of these mechanisms for BBB development. Furthermore, we demonstrated that HDAC inhibitors activate BBB gene cohorts expressed during BBB formation, suggesting a potential therapeutic intervention.
Transcriptional downregulation of key BBB genes in adult cortical ECs: evidence that distinct EC gene cohorts regulate BBB establishment versus maintenance
An intact, non-leaky BBB forms at E15.522. We hypothesized that the EC gene cohorts involved in BBB formation are expressed on E13.5, and 3-4 month-old adult CNS EC will express gene cohorts required for BBB maintenance. To test this, we defined transcriptomes of primary cortical ECs isolated from E13.5 and 3-4-month-old adults. mRNA-seq analysis revealed strong expression of EC genes including PECAM1, CDH5, and CLDN5 in comparison to perivascular cell types such as pericytes, neuronal and glial genes (S.Fig-1A). To define the gene cohorts responsible for BBB formation vs. BBB maintenance, we identified differentially expressed genes (DEGs) expressed in natural log (fold change) in E-13.5 relative to adult mice (Fig-1A,B). DEGs with a P-value < 0.05) were considered significant. In comparison to E13.5, 50% of genes were upregulated, 35% were downregulated in adults and 15% of genes were unchanged (Fig-1C).
GO enrichment analysis of the DEGs identified five functional categories: angiogenesis, cell-to-cell junction, transporters, extracellular matrix, and DNA binding transcription factors (Fig-1D). Except for transporters, the other categories were characterized by a greater number of downregulated vs. upregulated genes. The DEGs included important BBB genes (Fig-1E). For example, tight junction (TJ) gene CLDN1, CLDN5, BBB transporters MFSD2A, CAV1, and BBB-related transcription factors ZIC3, FOXF2, and SOX17 were differentially expressed (Fig-1E). The complete dataset is available on Geo under accession number: GSE214923 and presented in supplementary table-1. Overall the transcriptomic analysis identified EC gene cohorts that expressed during the formation or maintenance of the BBB.
We validated the differential expression of important BBB and related genes (e.g., CLDN1, CLDN5, MFSD2A, ZIC3, and SOX17) at E-13.5, E-17.5, P0, P7, and in the adult. This analysis revealed that CLDN1, MFSD2A, and ZIC3 expressions were significantly downregulated by E-17.5 and subsequent developmental stages (Fig-1F). Thus, the expression of these genes might be required for the establishment, but not maintenance, of the BBB. By contrast, adult expression of CLDN5 was significantly upregulated compared to other developmental stages and SOX17 was significant to other developmental stages except for P7(Fig-1F). Additionally, we validated the mRNA expression of CLDN11 and FOXF2 as well as the protein expression of CLDN1, CLDN5, and ZIC3 (S.Fig-B&C).
HDAC2 and PRC2 mediated transcriptional regulation of BBB genes
Histone deacetylases (HDACs) have critical roles in development and tissue homeostasis, and HDAC inhibitors are instructive experimental tools 23,24. To assess whether the transcriptional downregulation of BBB genes involves HDAC-dependent epigenetic repression, we treated adult ECs with a pan HDAC inhibitor trichostatin A (TSA) for 48 hours. TSA increased CLDN1 (Fig-2A) and ZIC3 (S.Fig-2A) expression relative to the control. Since there are four major HDAC classes, class-I (HDAC 1, 2, 3, and 8), class II (HDAC 4, 5, 6, 7, 9, 10), class III (SIRTs 1–7), and class IV (HDAC 11), we tested whether specific HDACs mediate the repression. The class-I HDAC inhibitor MS-275 significantly upregulated CLDN1, MFSD2A and ZIC3 while downregulated CLDN5 (Fig-2A, S.Fig-2A). No significant difference in CLDN1 mRNA expression was observed with class-II HDAC inhibitor (S.Fig-2A). In adult ECs, HDAC2 exhibited greater expression than other class I HDACs (Fig-2B). To analyze the HDAC2 function, we utilized siRNAs to downregulate HDAC1, HDAC2, or HDAC3 in adult ECs. Knockdown (KD) of HDAC2 significantly upregulated the repressed CLDN1, MFSD2A, and ZIC3 genes and reduced CLDN5 expression (Fig-2C). By contrast, HDAC1 and HDAC3 downregulation did not affect these genes (not shown). BBB genes analyzed above were selected based on their expression patterns and to represent important functional attributes of BBBs, such as tight junctions (CLDN1 and CLDN5), transporters (MFSD2A), and transcription factors (ZIC3). We used ChIP-qPCR to test whether HDAC2 directly regulates BBB gene expression in E13.5 and adult cortical EC contexts. Three primers were designed ((−)500, TSS & (+)500) spanning the 1 kb region on each side of the promoter. HDAC2 occupancy was detected in the (−)and(+) 500 regions of CLDN1 in adults with no significant enrichment in E13.5. ZIC3 showed occupancy at all three regions (1kb) in the adult stage with E13.5 showing enrichment in TSS only (Fig-2D,G &S.Fig2B). Conversely, HDAC2 occupied MFSD2A only at E13.5((−)500 & TSS), and in CLDN5 occupancy was detected at both E13.5 (1kb) and in adults ((−) & (+) 500). These results link HDAC2 to the developmental control of BBB genes (Fig-2D,G & S.Fig2B).
In our initial screening, we found that the PRC2 inhibitor DZNEP significantly increased CLDN1 expression in adult ECs (S.Fig-2A). To analyze the PRC2 function in this context, we downregulated the PRC2 subunit EZH2 from adult ECs. Compared to the control, EZH2 downregulation significantly increased CLDN1, ZIC3, and MFSD2A expression and decreased CLDN5 expression (Fig 2E). ChiP-qPCR of PRC2 subunit EED revealed EED occupancy at various regions of CLDN1, MFSD2A, and ZIC3 at E13.5 and in the adult (Fig2F,G & S.Fig-2). EED occupied CLDN5 at E13.5, but not in adult ECs. Together our data support a model in which HDAC2 and PRC2 are important determinants of EC BBB transcriptomes during BBB development.
Distinct histone modifications delineate the transcription program of BBB genes
Since HDAC2 and PRC2 are regulating multiple BBB genes we sought whether they share similar post-translational histone modifications. To examine this, we performed ChIP-qPCR of potentially involved histone marks, including the repressive marks H3K27me3, and H3K9me3 as well as the activating marks H3K4me3 and H3K9ac. We have scanned approximately 1kb genomic region surrounding the TSS of CLDN1, CLDN5, MFSD2A, ZIC3, and SOX17 in E13.5 and adults. H3K9me3 ChIP-qPCR on our selected BBB genes didn’t show significant binding in either stage (S.Fig-3A). We detected abundant enrichment of repressive histone mark H3K27me3 on CLDN1 (Fig-3A,C), MFSD2A, and ZIC3 (Fig-3C & S.Fig-3B,C) in adult ECs compared to E13.5. CLDN1, MFSD2A, and ZIC3 showed significant enrichment of H3K27me3 compared to IgG in E13.5 (Fig-3A & S.Fig-3B,C). While CLDN5 showed significant enrichment of H3K27me3 on 1kb region in both E13.5 and adults (Fig-3B). However, E13.5 showed abundant enrichment of H3K27me3 in the -500 region compared to adults (Fig-3B).
Active histone mark, H3K4me3 showed significantly increased enrichment on CLDN1 (TSS), MFSD2A (1KB), and ZIC3 (−500 and +500) at E13.5 compared to adult (Fig-3A and S.Fig-3B, C). CLDN1 (−)500 region didn’t show any significant binding for H3K4me3 in both stages, while the (+)500 region showed significant binding compared to IgG with no difference between E-13.5 and adult. ZIC3 TSS region showed significant enrichment of H3K4me3 in the adult compared to E-13.5(S.Fig-3). Supporting its abundant expression in adults, CLDN5 (1KB) showed significant enrichment of H3K4me3 in adults compared to E13.5. Another active histone mark H3K9ac showed significant enrichment on CLDN1 (TSS and +500) at E13.5 compared to the adult with no significant binding in the -500 region in both stages (Fig-3A & S.Fig-3). ZIC3 (TSS and +500) showed a significant binding for H3K9ac in E13.5 compared to the adult while the -500 region didn’t show any enrichment in both stages. MFSD2A showed significant enrichment of H3K9ac in the -500 and +500 regions at E-13.5 compared to adults. While the TSS region showed significant enrichment in adults compared to E-13.5. Histone modifications on SOX17 are shown in S.Fig-3D. These results indicate that BBB genes acquire a unique epigenetic signature during development.
HDAC2 activity is critical for the maturation of BBB, while PRC2 is dispensable
To examine the role of HDAC2 and PRC2 in BBB maturation, we knock out (KO) HDAC2 and PRC2 subunit EZH2 from ECs during embryonic development. To conditionally KO HDAC2 or EZH2 we used tamoxifen-inducible Cdh5(PAC)-CreERT2 mice. Tamoxifen was injected into the mother starting at E-12.5 and on alternate days until E-16.5 (Fig-4A & 4E). This allowed the KO of HDAC2 or EZH2 before the maturation of the BBB.
It was observed that HDAC2 and EZH2 KO embryos grew normally and were alive on the day of sacrifice E17.5. Significantly decreased mRNA expression of HDAC2 in the whole brain analysis confirmed the loss of HDAC2 (S.Fig-4). Compared to WT, HDAC2 ECKO pial vessels were dilated and showed increased angiogenesis (Fig-4B). BBB permeability was assessed in HDAC2 ECKO mice using a 70KD FITC-conjugated dextran tracer. The tracer remained confined inside the vessels of E17.5 WT embryos, supporting previous findings that the BBB matures by E-15.5 (Fig-4C). Conversely in HDAC2 ECKO, dextran leaked into the cortical parenchyma (Fig-4C). Green fluorescent intensity measurements confirmed the immature BBB in the HDAC2 ECKO (Fig-4D). A vascular analysis using angiotool determined that HDAC2 ECKO had a significantly higher vascularized brain area than W.T., indicating that HDAC2 plays a key role in angiogenesis (Fig-4D). While the pharmacological inhibition of class I HDAC using MS-275 in timed pregnant WT mice at E-13.5 showed significant leakage of tracer into the brain parenchyma at E-15.5 compared to vehicle-treated control (S.Fig-4A). The MS-275 treated embryos also showed a thin cortex compared to the control possibly due to the leakage of the drug into the brain which affects brain development (S.Fig-4A). These findings should be taken into account when considering the clinical use of MS-275 in a developing brain. In the whole brain mRNA analysis between the HDAC2 ECKO and WT showed significantly upregulated CLDN1 in HDAC2 ECKO (S.Fig-4B). No significant difference was observed in CLDN5, MFSD2A, and ZIC3 genes.
Compared to WT, at E-17.5 EZH2 ECKO showed dilated vessels with no evident increase in the pial vessel angiogenesis (Fig-4F). Confirming the loss of EZH2, mRNA analysis on the whole brain showed a significant reduction. BBB permeability assay showed a subtle leakage of 70KD fluorescent tracer into the brain parenchyma (Fig-4G). However, the fluorescence intensity analysis showed no significant difference (Fig-4H). Further, the mRNA analysis on the whole brain showed a significant decrease in EZH2 expression with no difference in the expression of CLDN1, MFSD2A, and CLDN5 (S.Fig-4C). Together, HDAC2 and EZH2 ECKO data suggest that HDAC2 is critical for BBB maturation, whereas PRC2 is dispensable or is a support mechanism that is required during later BBB development.
Despite Wnt pathway activity in the adult, Wnt target genes are epigenetically repressed
Wnt pathway has been shown to influence BBB gene expression 5. However, this pathway activity is reported to be minimum in adults15,17. In our transcriptomic analysis, 67% of Wnt signaling-related genes were downregulated in adults, while 33% were upregulated (Fig-5A). Interestingly, downstream Wnt targets genes including AXIN2, LEF1, and VEGFA have downregulated in adult ECs, while the upstream Wnt pathway components like FZD4/6, LRP5, and CTNNB1 were upregulated (Fig-5a). Since Wnt-regulated BBB genes such as CLDN1, and ZIC3 expression was also minimum in adults, we hypothesize that the Wnt pathway is still active in adult CNS ECs while Wnt target genes are epigenetically repressed.
To test this, we activated the Wnt pathway in E13.5 and adult ECs using identical concentrations of Wnt3a ligand or GSK3B inhibitor CHIR99021. mRNA analysis revealed that Wnt target genes AXIN2 and LEF1 can be significantly activated in E13.5 ECs when treated with Wnt3a or CHIR99021 while no significant activation was observed in adults (Fig-5B). Further, validating this finding, transcriptome analysis on adult ECs after Wnt3a treatment showed only activation of one Wnt target gene CD44 (Fig-5C). Intriguingly, the Wnt3a treatment downregulated 16 Wnt-related genes (Fig-5C). Next using, immunohistochemistry of β-catenin in adult control and CHIR 99021 treated ECs we demonstrated that Wnt pathway activation could translocate or stabilize the Wnt transducer β-catenin into the nucleus (Fig-5D). These results indicate that the Wnt pathway is active in adult CNS ECs.
Next, we investigated whether Wnt target genes are epigenetically repressed. Wnt target genes AXIN2 and LEF1 were significantly upregulated in adult CNS ECs when treated with HDAC2 or EZH2 siRNA, MS-275, and in HDAC2 ECKO mutants (Fig-5E & S.F-5A,B). Furthermore, the AXIN2 promoter in adults showed significantly increased occupancy of HDAC2, repressive histone mark H3K27me3 in adults while active histone marks H3K4me3 and H3K9ac were significantly reduced compared to E-13.5 as analyzed by ChIP-qPCR (Fig 5F). LEF1 also showed a similar repressive histone modification (Fig-5C). Further MS-275 and LiCl (Wnt agonist) treatment increase the AXIN2 protein expression in adult cortical vessels but not by just LiCl (S.Fig-5D). Combined, our data explain the mechanism behind the low Wnt pathway in adult CNS ECs.
Low Wnt signaling epigenetically modifies the BBB genes to achieve BBB maturation
We investigated the relevance of the low Wnt pathway to BBB development. To this, we treated primary E13.5 cortical ECs with LF3 (inhibits the interaction of β-catenin and TCF4) for 48 hours. LF3 activity was confirmed by reduced mRNA expression of the Wnt target gene AXIN2 (Fig-6A). LF3 treatment induces adult/BBB maintenance type gene expression patterns in E13.5 with a significant decrease in CLDN1, ZIC3, and MFSD2A expression and an increase in CLDN5 and SOX17 expression (Fig-6A). Another Wnt inhibitor IWR-1-endo also showed similar results (not shown) while β-catenin siRNA KD showed a similar gene expression pattern for AXIN2, CLDN1, ZIC3, and MFSD2A and no difference in CLDN5 (S.Fig-6A).
To determine whether Wnt pathway inhibition induces epigenetic modifications on its target genes and BBB genes, we performed Chip-qPCR analysis of HDAC2, EED, and histone mark H3K27me3, H3K4me3, and H3K9ac on the promoter of Wnt target genes AXIN2, and LEF1, BBB genes CLDN1, CLDN5, MFSD2A, and ZIC3. An increased HDAC2 occupancy was observed on the promoter of AXIN2, LEF1, CLDN1, and MFSD2A when E13.5 ECs were treated with LF3 (Fig-6B & S.Fig-6B). ZIC3 and CLDN5 showed no significant difference (not shown). EED and histone mark H3K27me3 showed an increased enrichment on the AXIN2 while LEF1 showed increased enrichment for H3K27me3 in the promoter after LF3 treatment (Fig-6B & S.Fig-6B) with no difference in other genes analyzed (not shown). CLDN1 and MFSD2A showed a significant decrease in the enrichment of active histone mark H3K4me3 after treatment with LF3, whereas CLDN5 showed a significant increase (Fig-6B). Another active histone mark H3K9ac showed significantly increased enrichment on CLDN5 (Fig-6B) with no difference in the promoter of other genes analyzed (not shown).
We then investigate the significance of the physiological reduction of Wnt signaling on BBB maturation. For this we use Ctnnb1lox(ex3)+/+; Cdh5-CreERT2 mice which are widely used to attain the inducible EC specific β-catenin gain of function (GOF) (Fig-6C). Upon tamoxifen treatment exon 3 of CTNNB1 (encoding β-catenin) will be deleted leading to the expression of a stabilized form of β-catenin protein and thereby constitutive activation of canonical Wnt signaling (Fig6C). Tamoxifen was injected into the pregnant mother as explained in section 2.4. At E-17.5 β-catenin GOF embryos showed significantly increased pial angiogenesis (Fig-6D) compared to WT. BBB permeability assay using 70KD FITC dextran revealed immature BBB in β-catenin GOF (Fig-6E & F). Confirming this result pharmacological activation of Wnt signaling by Wnt agonist LiCl also showed a significant BBB leakage compared to the control (S.Fig-6C). Together, our data suggest that a low Wnt pathway supports BBB maturations by epigenetically modifying BBB genes.
Class-I HDAC inhibitor treatment of adult CNS ECs re-establishes gene cohorts required for BBB formation
To obtain a detailed picture of CNS EC genes regulated after MS-275 treatment we performed an mRNA-seq analysis of MS-275-treated adult ECs and compared this with the control. MS-275 treatment upregulated 48% of genes, downregulated 34% of genes, and did not alter the expression of 18% of genes. Differentially expressed genes were grouped into six relevant categories and all the categories showed a significant no.of differentially regulated genes (Fig-7A). Fig-7B illustrates the potential of MS-275 in regaining BBB, angiogenesis, Wnt, and transcription factors that are differentially regulated during development.
We next assessed whether the gene expression changes acquired by adult ECs following MS-275 treatment were reversible. To this end, we treated adult ECs with MS-275 for 48 hours and collected ECs at 48 hours of treatment and 7 days after treatment. As previously shown, CLDN1 was significantly upregulated and CLDN5 was significantly downregulated after 48 hours (Fig-7C). While the expression of CLDN1 and CLDN5 in adult ECs returned to normal in ECs collected 7 days after the treatment (Fig-7C).
Finally, we examined if human vessels show similar reactivation when treated with MS-275. To this end, the human brain vessels collected from epilepsy surgery irrespective of age and gender. We tested three genes with MS-275 treatment: CLDN1, CLDN5, and AXIN2. We found that MS-275 treatment significantly activates the expression of CLDN1 and AXIN2 compared to the control, with no difference in CLDN5 expression (Fig-7D).
The mechanisms that create and maintain the BBB are vitally important, but they are poorly understood. We identified EC gene cohorts that differentially support BBB formation and maintenance, describe the genetic and signaling mechanisms that establish the BBB, and present an attractive strategy to activate gene cohorts involved in BBB formation that may promote BBB repair.
MFSD2A is required for BBB formation22, and the transcription factors ZIC3 and FOXF2 can induce BBB markers even in peripheral ECs5. Our transcriptomic analysis revealed that CLDN1, MFSD2A, ZIC3, and FOXF2 were significantly expressed in E13.5 compared to adults, validating their requirement in BBB formation. Increased levels of CLDN5, PECAM, BCG1, and SOX17 in adult CNS ECs point to its significance in BBB maintenance. The results of our study agree with those of prior gene expression studies, but provide new concepts regarding downstream mechanisms governing BBB gene expression. Although a high level of purity is achieved in primary EC culture, we cannot exclude the fact that transcripts from other cell populations may be identified.
It is not known how BBB gene transcription is regulated. In CNS ECs, HDAC2 and PRC2 directly regulate the transcription of important BBB genes including CLDN1, CLDN5, MFSD2A, and ZIC3. While HDAC2 and PRC2 commonly occupy repressed genes, we detected them at BBB genes in both active and repressive states. This result is consistent with the established dual transcriptional role of this epigenetic regulators25-32. Furthermore, we present evidence that HDAC2 is required for the formation of a functional BBB and induction of anti-angiogenic signals. Even though the loss of vascular integrity and lethality at E-13.5 was reported in non-inducible conditional EZH2 KO using Tie2 Cre-mouse 33, loss of EZH2 from E-13.5 did not significantly affect BBB permeability. These results indicate that, during the differentiation phase, HDAC2 initiates the transcriptional control, and PRC2 functions in a supporting mechanism. However, the KD of these regulators from adult CNS ECs induces a similar gene expression pattern indicating the possibility that PRC2 facilitates the epigenetic modification during later BBB development and maintenance.
Since we did not detect H3K9me3, the repression of genes involved in BBB formation is mainly mediated through H3K27me3. On varying abundance repressive histone mark H3K27me3 and active histone mark, H3K4me3 was detected in our selected BBB genes, suggesting that the promoter is modified by repressive and activating histone methylation marks. Bivalent histone states can correlate with genes transcribed at low levels, suggesting that these genes are poised for activation34. In addition, we detected H3K9ac at the active and repressed BBB genes. Bivalent promoters can harbor H3K9ac. Thus, among the five BBB genes analyzed, each gene exhibits a different epigenetic signature, defined by histone acetylation and methylation. These results systematically demonstrate the complexity of epigenetic regulation in BBB genes and the evidence of unique epigenetic signatures. The data presented here is not completely representing all the histone modifications on BBB genes, as other important histone modifications such as H3K27ac and H3K14ac have not been examined.
It is still unknown what mechanism confers low Wnt signaling in adult CNS ECs. Our results demonstrate that Wnt pathway components are still active in adult CNS ECs, yet the Wnt target genes are epigenetically inactive. Thus, in the adult CNS ECs, Wnt pathway activation permits stabilized β-catenin to enter the nucleus. Whether this nuclear β-catenin binds to Wnt target genes is unknown. Nevertheless, we demonstrated that HDAC2 and PRC2 repress Wnt target genes. Other studies have revealed that the basal or minimal Wnt pathway maintains adult BBB integrity, but also its activation prevents stroke-induced BBB damage. This also suggests the possibility of switching Wnt-regulated genes during development. Our transcriptomic analysis revealed the Wnt-regulated adult CNS EC genes associated with BBB (Table S2).
It is unknown how Wnt regulates BBB genes. We demonstrated that inhibiting the active Wnt pathway at E13.5 can induce the BBB gene expression pattern associated with BBB maintenance. We demonstrate that the low Wnt pathway in E13.5 causes epigenetic modifications on BBB genes mediated by HDAC2. However, the link between Wnt and epigenetic mechanisms is unclear. Since inhibiting β-catenin induce the aforementioned results, in principle, it may be mediated through β-catenin. In support of this model, β-catenin GOF during BBB development prevents the maturation of BBB and inhibits the differentiation of angiogenic vessels which results in increased brain vascularization. Similar morphological characteristics between HDAC2 ECKO and β-catenin GOF point to its close association.
Wnt signaling in CNS ECs is believed to require Wnt7a and Wnt7b produced by neural progenitors35-38. Our transcriptomic data revealed significant expression of Wnt7a in E13.5 ECs with null expression in adults. Interestingly, adult CNS ECs showed significant activation of Wnt7a mRNA with MS-275 treatment (S.Fig7A). Moreover, β-catenin KD and GOF affect the expression of Wnt7a, and β -catenin staining after MS-275 treatment showed localization of β-catenin to the nucleus. These data indicate an innate mechanism of ECs to regulate the Wnt pathway.
A natural consequence of targeting epigenetic regulators or the Wnt pathway is that these mechanisms regulate numerous genes. Our mRNA sequencing results revealed that class-1 HDAC inhibitor treatment regulates EC genes associated with key functions including angiogenesis, barriergenesis, and Wnt signaling. Epidrugs are attractive therapeutics since epigenetic changes are reversible, with the potential to reestablish function after treatment. Our results support this, as the expression of CLDN1 and CLDN5 returns to the normal level after 7 days of treatment. Furthermore, our results illustrate the potential of MS-275 to reinstate the developmental characteristics in mice and partially in human adult CNS ECs.
We thank Ralf Adams (Max Planck Institute for Molecular Biomedicine) and Ondine Cleaver (The University of Texas Southwestern) for providing the Cdh5-CreERT2 mice. This work was also supported by American Heart Association Career Development Award (18CDA34110036), NIH grant R01 to PKT (R01NS121339), and UT Health startup funds.
JS and ST performed the experiments and analyzed the data under the supervision of PKT. IEM performed western blots and analyzed RNA-seq data. PKT conceived and supervised the project, designed, and performed experiments, interpreted the data, and wrote the manuscript. HZ primarily contributed to the idea of Wnt target gene repression in adult cortical ECs. EHB provided critical comments on the manuscript and ChIP data. NT provide the human cortical vessels. AH and HL analyzed the RNA-seq data. All the authors critically commented on the manuscript.
- 1The blood-brain barrier in health and disease: Important unanswered questionsJ Exp Med 217
- 2Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cellsElife 7
- 3The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cellsPLoS One 5
- 4Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain BarrierCirc Res 124:511–525
- 5Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levelsSci Signal 10
- 6The molecular hallmarks of epigenetic controlNat Rev Genet 17:487–500
- 7Histone acetylation in chromatin structure and transcriptionNature 389:349–352
- 8HDACs and HDAC Inhibitors in Cancer Development and TherapyCold Spring Harb Perspect Med 6
- 9HDAC and HDAC Inhibitor: From Cancer to Cardiovascular DiseasesChonnam Med J 52:1–11
- 10Medicinal chemistry updates of novel HDACs inhibitors (2020 to present)Eur J Med Chem 227
- 11Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste proteinGenes Dev 16:2893–2905
- 12Role of histone H3 lysine 27 methylation in Polycomb-group silencingScience 298:1039–1043
- 13Tazemetostat: EZH2 InhibitorJ Adv Pract Oncol 13:158–163
- 14Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesisProc Natl Acad Sci U S A 106:641–646
- 15Wnt/beta-catenin signaling controls development of the blood-brain barrierJ Cell Biol 183:409–417
- 16Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculatureScience 322:1247–1250
- 17Radial glial neural progenitors regulate nascent brain vascular network stabilization via inhibition of Wnt signalingPLoS Biol 11
- 18Endothelial Wnt/beta-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expressionJ Exp Med 209:1611–1627
- 19Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculaturesElife 8
- 20Wnt signaling mediates acquisition of blood-brain barrier properties in naive endothelium derived from human pluripotent stem cellsElife 10
- 21Endothelial beta-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System HomeostasisCirculation 133:177–186
- 22Mfsd2a is critical for the formation and function of the blood-brain barrierNature 509:507–511
- 23Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitorsClin Epigenetics 4
- 24Histone deacetylase inhibitors: a chemical genetics approach to understanding cellular functionsBiochim Biophys Acta 1799:717–725
- 25Polycomb-dependent H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelityMol Cell 53:49–62
- 26Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNANat Struct Mol Biol 24:1028–1038
- 27Histone Deacetylases Positively Regulate Transcription through the Elongation MachineryCell Rep 13:1444–1455
- 28Transcription-dependent association of HDAC2 with active chromatinJ Cell Physiol 233:1650–1657
- 29CRISPR-mediated HDAC2 disruption identifies two distinct classes of target genes in human cellsPLoS One 12
- 30Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genesCell 138:1019–1031
- 31Negative and positive regulation of gene expression by mouse histone deacetylase 1Mol Cell Biol 26:7913–7928
- 32PRC2 binds active promoters and contacts nascent RNAs in embryonic stem cellsNat Struct Mol Biol 20:1258–1264
- 33Ezh2-mediated repression of a transcriptional pathway upstream of Mmp9 maintains integrity of the developing vasculatureDevelopment 141:4610–4617
- 34A bivalent chromatin structure marks key developmental genes in embryonic stem cellsCell 125:315–326
- 35A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt BioavailabilityCell Rep 25:339–349
- 36A molecular mechanism for Wnt ligand-specific signalingScience 361
- 37Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124Science 330:985–989
- 38Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier RegulationNeuron 95:1056–1073