Egr2-guided histone H2B monoubiquitination is required for peripheral nervous system myelination.

Schwann cells are the nerve ensheathing cells of the peripheral nervous system. Absence, loss and malfunction of Schwann cells or their myelin sheaths lead to peripheral neuropathies such as Charcot-Marie-Tooth disease in humans. During Schwann cell development and myelination chromatin is dramatically modified. However, impact and functional relevance of these modifications are poorly understood. Here, we analyzed histone H2B monoubiquitination as one such chromatin modification by conditionally deleting the Rnf40 subunit of the responsible E3 ligase in mice. Rnf40-deficient Schwann cells were arrested immediately before myelination or generated abnormally thin, unstable myelin, resulting in a peripheral neuropathy characterized by hypomyelination and progressive axonal degeneration. By combining sequencing techniques with functional studies we show that H2B monoubiquitination does not influence global gene expression patterns, but instead ensures selective high expression of myelin and lipid biosynthesis genes and proper repression of immaturity genes. This requires the specific recruitment of the Rnf40-containing E3 ligase by Egr2, the central transcriptional regulator of peripheral myelination, to its target genes. Our study identifies histone ubiquitination as essential for Schwann cell myelination and unravels new disease-relevant links between chromatin modifications and transcription factors in the underlying regulatory network.

Cooperation, cis-interactions, versatility and evolutionary plasticity of multiple cis-acting elements underlie krox20 hindbrain regulation.

Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes.

Egr2 enhances insulin resistance via JAK2/STAT3/SOCS-1 pathway in HepG2 cells treated with palmitate.

Insulin resistance is generally responsible for the pathogenesis of type 2 diabetes mellitus (T2DM). Early growth response proteins-2 (Egr2) has been reported to be able to increase the expression of the suppressors of cytokine signaling-1 (SOCS-1), and impair insulin signaling pathway through suppression of insulin receptor substrates (IRS), including IRS-1 and IRS-2. However, whether Egr2 is directly involved in the development of insulin resistance, and how its potential contributions to insulin resistance still remain unknown. Here, our present investigation found that the expression levels of Egr2 were up-regulated when insulin resistance occurs, and knockdown of Egr2 abolished the effect of insulin resistance in HepG2 cells induced with palmitate (PA). Importantly, inhibition of Egr2 decreased the expression of SOCS-1 as well as reduced phosphorylation of JAK2 and STAT3. And, our data indicated that silencing of Egr2 accelerated hepatic glucose uptake and reversed the impaired lipid metabolism upon insulin resistance. In summary, the present study confirms that Egr2 could deteriorate insulin resistance via the pathway of JAK2/STAT3/SOCS-1 and may shed light on resolving insulin resistance and further the pathogenesis of T2DM.

EGR2 is critical for peripheral naïve T-cell differentiation and the T-cell response to influenza.

Early growth response 2 (EGR2) transcription factor negatively regulates T-cell activation, in contrast to the positive regulation of this process by EGR1. Here, we unexpectedly found that EGR2 promotes peripheral naïve T-cell differentiation, with delayed T-cell receptor-induced proliferation in naïve T cells from Egr2 conditional knockout (CKO) mice and decreased production of IFN-γ, IL-4, IL-9, and IL-17A in cells subjected to T-helper differentiation. Moreover, genes that promote T-cell activation, including Tbx21 and Notch1, had decreased expression in Egr2 CKO T cells and are direct EGR2 target genes. Following influenza infection, Egr2 CKO mice had delayed viral clearance, more weight loss, and more severe pathological changes in the lung than did WT and Egr1 KO mice, with decreased production of effector cytokines, increased infiltration of antigen-specific memory-precursor CD8(+) T cells, and lower numbers of lung-resident memory CD8(+) T cells. Thus, unexpectedly, EGR2 can function as a positive regulator that is essential for naïve T-cell differentiation and in vivo T-cell responses to a viral infection.

Early Growth Response Genes 2 and 3 Regulate the Expression of Bcl6 and Differentiation of T Follicular Helper Cells.

T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GCs), and Tfh differentiation requires the function of B cell lymphoma 6 (BCL6). We have now discovered that early growth response gene 2 (EGR2) and EGR3 directly regulate the expression of Bcl6 in Tfh cells, which is required for their function in regulation of GC formation. In the absence of EGR2 and -3, the expression of BCL6 in Tfh cells is defective, leading to impaired differentiation of Tfh cells, resulting in a failure to form GCs following virus infection and defects in production of antiviral antibodies. Enforced expression of BCL6 in EGR2/3-deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of EGR2/3 that is important for Tfh cell development and Tfh cell-mediated B cell immune responses.

Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiation.

The oncogenic cluster miR-17-92 encodes seven related microRNAs that regulate cell proliferation, apoptosis and development. Expression of miR-17-92 cluster is decreased upon cell differentiation. Here, we report a novel mechanism of the regulation of miR-17-92 cluster. Using transgenic PU.1(-/-) myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant Egr2 which, in turn, directly represses miR-17-92 expression by recruiting histone demethylase Jarid1b leading to histone H3 lysine K4 demethylation within the CpG island at the miR-17-92 promoter. Conversely, Egr2 itself is targeted by miR-17-92, indicating existence of mutual regulatory relationship between miR-17-92 and Egr2. Furthermore, restoring EGR2 levels in primary acute myeloid leukaemia blasts expressing elevated levels of miR-17-92 and low levels of PU.1 and EGR2 leads to downregulation of miR-17-92 and restored expression of its targets p21CIP1 and BIM. We propose that upon macrophage differentiation PU.1 represses the miR-17-92 cluster promoter by an Egr-2/Jarid1b-mediated H3K4 demethylation mechanism whose deregulation may contribute to leukaemic states.

Early growth response gene-2 controls IL-17 expression and Th17 differentiation by negatively regulating Batf.

Early growth response gene (Egr)-2 is important for the maintenance of T cell homeostasis and controls the development of autoimmune disease. However, the underlying mechanisms are unknown. We have now discovered that Egr-2, which is induced by TGF-ß and IL-6, negatively regulates the expression of IL-17, but not IL-2 or IFN-γ, in effector T cells. In the absence of Egr-2, CD4 T cells produce high levels of Th17 cytokines, which renders mice susceptible to experimental autoimmune encephalomyelitis induction. T cells lacking Egr-2 show increased propensity for Th17, but not Th1 or Th2, differentiation. Control of IL-17 expression and Th17 differentiation by Egr-2 is due to inhibition of Batf, a transcription factor that regulates IL-17 expression and Th17 differentiation. Egr-2 interacts with Batf in CD4 T cells and suppresses its interaction with DNA sequences derived from the IL-17 promoter, whereas the activation of STAT3 and expression of retinoic acid-related orphan receptor γt are unchanged in Th17 cells in the absence of Egr-2. Thus, Egr-2 plays an important role to intrinsically control Th17 differentiation. We also found that CD4 T cells from multiple sclerosis patients have reduced expression of Egr-2 and increased expression of IL-17 following stimulation with anti-CD3 in vitro. Collectively, our results demonstrate that Egr-2 is an intrinsic regulator that controls Th17 differentiation by inhibiting Batf activation, which may be important for the control of multiple sclerosis development.

Significant pathways detection in osteoporosis based on the bibliometric network.

BACKGROUND: Osteoporosis is a significant public health issue worldwide. The underlying mechanism of osteoporosis is an imbalance between bone resorption and bone formation. However, the exact pathology is still unclear, and more related genes are on demand. AIM: Here, we aim to identify the differentially expressed genes in osteoporosis patients and control. MATERIALS AND METHODS: Biblio-MetReS, a tool to reconstruct gene and protein networks from automated literature analysis, was used for identifying potential interactions among target genes. Relevant signaling pathways were also identified through pathway enrichment analysis. RESULTS: Our results showed that 56 differentially expressed genes were identified. Of them, STAT1, CXCL10, SOCS3, ADM, THBS1, SOD2, and ERG2 have been demonstrated involving in osteoporosis. Further, a bibliometric network was constructed between DEGs and other genes through the Biblio-MetReS. CONCLUSIONS: The results showed that STAT1 could interact with CXCL10 through Toll-like receptor signaling pathway and Chemokine signaling pathway. STAT1 interacted with SOCS3 through JAK/STAT pathway.

CD4+CD25-LAG3+ regulatory T cells controlled by the transcription factor Egr-2.

Regulatory T cells (Tregs) are engaged in the maintenance of immunological self-tolerance and immune homeostasis. IL-10 has an important role in maintaining the normal immune state. Here, we show that IL-10-secreting Tregs can be delineated in normal mice as CD4(+)CD25(-)Foxp3(-) T cells that express lymphocyte activation gene 3 (LAG-3), an MHC-class-II-binding CD4 homolog. Although approximately 2% of the CD4(+)CD25(-) T cell population consisted of CD4(+)CD25(-)LAG3(+) T cells in the spleen, CD4(+)CD25(-)LAG3(+) T cells are enriched to approximately 8% in the Peyer's patch. They are hypoproliferative upon in vitro antigenic stimulation and suppress in vivo development of colitis. Gene expression analysis reveals that CD4(+)CD25(-)LAG3(+) Tregs characteristically express early growth response gene 2 (Egr-2), a key molecule for anergy induction. Retroviral gene transfer of Egr-2 converts naïve CD4(+) T cells into the IL-10-secreting and LAG-3-expressing phenotype, and Egr-2-transduced CD4(+) T cells exhibit antigen-specific immunosuppressive capacity in vivo. Unlike Foxp3(+) natural Tregs, high-affinity interactions with selecting peptide/MHC ligands expressed in the thymus do not induce the development of CD4(+)CD25(-)LAG3(+) Tregs. In contrast, the number of CD4(+)CD25(-)LAG3(+) Tregs is influenced by the presence of environmental microbiota. Thus, IL-10-secreting Egr-2(+)LAG3(+)CD4(+) Tregs can be exploited for the control of peripheral immunity.

Krox20 inactivation in the PNS leads to CNS/PNS boundary transgression by central glia.

CNS/PNS interfaces constitute cell boundaries, since they delimit territories with different neuronal and glial contents. Despite their potential interest in regenerative medicine, the mechanisms restricting oligodendrocytes and astrocytes to the CNS, and Schwann cells to the PNS in mammals are not known. To investigate the involvement of peripheral glia and myelin in the maintenance of the CNS/PNS boundary, we have first made use of different mouse mutants. We show that inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, results in transgression of the CNS/PNS boundary by astrocytes and oligodendrocytes and in myelination of nerve root axons by oligodendrocytes. In contrast, such migration does not occur with the Trembler(J) mutation, which prevents PNS myelination without affecting Krox20 expression. Altogether these data suggest that maintenance of the CNS/PNS boundary requires a new Krox20 function separable from myelination control. Finally, we have analyzed a human patient affected by a congenital amyelinating neuropathy, associated with the absence of the KROX20 protein in Schwann cells. In this case, the nerve roots were also invaded by oligodendrocytes and astrocytes. This indicates that transgression of the CNS/PNS boundary by central glia can occur in pathological situations in humans and suggests that the underlying mechanisms are common with the mouse.

[Krox20 inactivation in the PNS leads to CNS/PNS boundary transgression by central glia]. / Système nerveux central, système nerveux pèriphérique: comment maintenir la frontière?

CNS/PNS interfaces constitute cell boundaries, defining territories with different neuronal and glial contents. Despite their potential implications for regenerative medicine, the mechanisms that restrict oligodendrocytes and astrocytes to the CNS and Schwann cells to the PNS are not known in mammals. To investigate the involvement of peripheral glia and myelin in CNS/PNS boundary maintenance, we first studied mutant mice. We found that inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, resulted in CNS/PNS boundary transgression by astrocytes and oligodendrocytes, and in myelination of nerve root axons by oligodendrocytes. In contrast, no such migration was observed in mice with the Trembler(J) mutation, which prevents PNS myelination without affecting Krox20 expression. These findings suggest that CNS/PNS boundary maintenance requires a new Krox20 function independent of myelination control. We also examined a patient with congenital amyelinating neuropathy, whose Schwann cells lack KROX20 protein. Interestingly, the patient's nerve roots were also invaded by oligodendrocytes and astrocytes, indicating that CNS/PNS boundary transgression by central glia can occur in human pathological situations and that the underlying mechanisms are the same as in mutant mice.

Analysis of peripheral nerve expression profiles identifies a novel myelin glycoprotein, MP11.

The myelin sheath insulates axons and allows for rapid salutatory conduction in the nervous system of all vertebrates. The formation of peripheral myelin requires expression of the transcription factor Egr2, which is responsible for inducing such essential myelin-associated genes as Mpz, Mbp, Pmp22, and Mag. Using microarray analysis to compare gene expression patterns in peripheral nerve during development, during remyelination after nerve injury, and in a congenital hypomyelinating mouse model, we identified an evolutionarily conserved novel component of myelin called Mp11 (myelin protein of 11 kDa). The Mp11 genomic locus contains multiple conserved Egr binding sites, and Mp11 induction is regulated by the expression of Egr2. Similar to other Egr2-dependent genes, it is induced during developmental myelination and remyelination after nerve injury. Mp11 is a glycoprotein expressed preferentially in the myelin of the peripheral nervous system versus CNS and is specifically localized to the Schmidt-Lanterman incisures and paranodes of peripheral nerve. The Mp11 protein contains no identifiable similarity to other known protein domains or motifs. However, like other myelin genes, strict Mp11 expression levels are a requirement for the in vitro myelination of DRG neurons, indicating that this previously uncharacterized gene product is a critical component of peripheral nervous system myelin.

[Boundary cap cells--a nest of neural stem cells in the peripheral nervous system]. / Les cellules des capsules frontières--une niche de cellules souches neurales dans le système nerveux périphérique.

The peripheral nervous system (PNS) is formed by neural crest cells (NCC) that migrate out of the neural tube in early mid-gestation. NCC give rise to most components of the PNS, including sensory neurons, glial satellite cells and Schwann cells. Neural crest cells also give rise to another type of PNS cell population named boundary cap (BC) cells, that form clusters at the surface of the neural tube, at entry and exit points of peripheral nerve roots. Using various genetic tools we were able to trace BC cell progeny during development and to ablate them in vivo. This revealed a previously unsuspected function of BC cells: they are required to maintain the integrity of the spinal cord motor column as, in their absence, motor neurons translocate their cell bodies along their axons into the periphery. In addition, we found that trunk BC-derived cells migrated along peripheral axons and colonized spinal nerve roots and dorsal root ganglia (DRG). All Schwann cell precursors occupying the dorsal roots were derived from BC cells. In the DRG, BC-derived cells were the progenitors of both neurons (mainly nociceptive afferents) and satellite cells. These unexpected observations indicate that BC cells constitute a source of peripheral nervous system (PNS) components that, after the major neural crest ventrolateral migratory stream, feed a secondary wave of migration to the PNS.

The EGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen-specific CD8+ T cells in the tumor microenvironment.

Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous antitumor response, immune regulatory pathways can subvert the effector phase and enable tumor escape. Negative regulatory pathways include extrinsic suppression mechanisms, but also a T cell-intrinsic dysfunctional state. A more detailed study has been hampered by a lack of cell surface markers defining tumor-specific dysfunctional TILs, and PD-1 alone is not sufficient. Recently, we identified the transcription factor Egr2 as a critical component in controlling the anergic state in vitro. In this study, we show that the Egr2-driven cell surface proteins LAG-3 and 4-1BB can identify dysfunctional tumor antigen-specific CD8+ TIL. Co-expression of 4-1BB and LAG-3 was seen on a majority of CD8+ TILs, but not in lymphoid organs. Functional analysis revealed defective IL-2 and TNF production yet retained expression of IFN-γ and regulatory T cell-recruiting chemokines. Transcriptional and phenotypic characterization revealed coexpression of multiple additional co-stimulatory and co-inhibitory receptors. Administration of anti-LAG-3 plus anti-4-1BB mAbs was therapeutic against tumors in vivo, which correlated with phenotypic normalization. Our results indicate that coexpression of LAG-3 and 4-1BB characterize dysfunctional T cells within tumors, and that targeting these receptors has therapeutic utility.

Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells.

Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies.

Functional Role of γ-Crystallin N in the Auditory Hindbrain.

γ-crystallins are major components of the vertebrate lens but show expression in other tissues as well. Their extralenticular functions remain so far unclear. Here, we explored such roles in the rodent superior olivary complex in which previous analysis demonstrated developmentally regulated expression of Crygd, Cryge and Crygn. Immunohistochemistry with novel antibodies against Crygd/e and Crygn indicate that expression of Crygd/e was moderate and varied between the perinatal superior olivary complex of mice, rats, and gerbils. Crygn-immunoreactivity was more robust and consistently highest in the medial nucleus of the trapezoid body, but also present in other nuclei of the superior olivary complex. To analyze the function of Crygn in the auditory hindbrain, we used a Crygn allele with a floxed exon 2. Upon pairing with Egr2::Cre mice, exon 2, encoding the first two greek key motifs of Crygn, was deleted in the developing auditory hindbrain. Anatomical analysis of these mice revealed a 20% volume reduction in the medial nucleus of the trapezoid body and a 7% reduction in the lateral superior olive at postnatal day 25. This was due to cell loss between postnatal days 4 and 25, whereas cell size was unaffected. Auditory brainstem responses showed normal threshold but a significant increase in the amplitude of wave IV. Crygn is hence required for postmigratory survival and proper function of auditory hindbrain neurons. These results ascertain for the first time an essential extralenticular role for γ-crystallins in vivo.

Genetic and pharmacological inactivation of adenosine A2A receptor reveals an Egr-2-mediated transcriptional regulatory network in the mouse striatum.

The adenosine A2A receptor (A2AR) is highly expressed in the striatum, where it modulates motor and emotional behaviors. We used both microarray and bioinformatics analyses to compare gene expression profiles by genetic and pharmacological inactivation of A2AR and inferred an A2AR-controlled transcription network in the mouse striatum. A comparison between vehicle (VEH)-treated A2AR knockout (KO) mice (A2AR KO-VEH) and wild-type (WT) mice (WT-VEH) revealed 36 upregulated genes that were partially mimicked by treatment with SCH-58261 (SCH; an A2AR antagonist) and 54 downregulated genes that were not mimicked by SCH treatment. We validated the A2AR as a specific drug target for SCH by comparing A2AR KO-SCH and A2AR KO-VEH groups. The unique downregulation effect of A2AR KO was confirmed by comparing A2AR KO-SCH with WT-SCH gene groups. The distinct striatal gene expression profiles induced by A2AR KO and SCH should provide clues to the molecular mechanisms underlying the different phenotypes observed after genetic and pharmacological inactivation of A2AR. Furthermore, bioinformatics analysis discovered that Egr-2 binding sites were statistically overrepresented in the proximal promoters of A2AR KO-affected genes relative to the unaffected genes. This finding was further substantiated by the demonstration that the Egr-2 mRNA level increased in the striatum of both A2AR KO and SCH-treated mice and that striatal Egr-2 binding activity in the promoters of two A2AR KO-affected genes was enhanced in A2AR KO mice as assayed by chromatin immunoprecipitation. Taken together, these results strongly support the existence of an Egr-2-directed transcriptional regulatory network controlled by striatal A2ARs.

Neural tube patterning by Krox20 and emergence of a respiratory control.

Recent data begin to bridge the gap between developmental events controlling hindbrain neural tube regional patterning and the emergence of breathing behaviour in the fetus and its vital adaptive function after birth. In vertebrates, Hox paralogs and Hox-regulating genes orchestrate, in a conserved manner, the transient formation of developmental compartments in the hindbrain, the rhombomeres, in which rhythmic neuronal networks of the brainstem develop. Genetic inactivation of some of these genes in mice leads to pathological breathing at birth pointing to the vital importance of rhombomere 3 and 4 derived territories for maintenance of the breathing frequency. In chick embryo at E7, we investigated neuronal activities generated in neural tube islands deriving from combinations of rhombomeres isolated at embryonic day E1.5. Using a gain of function approach, we reveal a role of the transcription factor Krox20, specifying rhombomeres 3 and 5, in inducing a rhythm generator at the parafacial level of the hindbrain. The developmental genes selecting and regionally coordinating the fate of CNS progenitors may hold further clues to conserved aspects of neuronal network formation and function. However, the most immediate concern is to take advantage of early generated rhythmic activities in the hindbrain to pursue their downstream cellular and molecular targets, for it seems likely that it will be here that rhythmogenic properties will eventually take on a vital role at birth.

[The functions of CD4+CD25-LAG3+ regulatory T cells and Egr2 in the regulation of autoimmunity].

Regulatory T cells (Treg) are important mechanisms that regulate autoimmunity and CD4+CD25+Foxp3+ regulatory T cells (CD25+Treg) have been extensively investigated. Recently, we have identified CD4+CD25-LAG3+ regulatory T cells (LAG3+Treg) that express an anergy associated transcription factor Egr2. Egr2 regulates IL-10 production in response to IL-27, and Egr2-deficiency in T cells and B cells results in systemic autoimmunity with increased IL-17 production. Moreover, addition of Egr3 deficiency to Egr2-deficient mice significantly accelerates systemic autoimmunity without functional impairment of CD25+Treg, indicating cooperative autoimmune-regulation by Egr2 and Egr3. The linkage between Egr2 and systemic autoimmunity is also suggested by the fact that stimulation with Ly108, a candidate lupus susceptibility gene in lupus-prone NZM2410 mice, induces Egr2 expression in T cells. Collectively, LAG3+Treg and Egr2 are the unique regulators of autoimmunity and further examination may help to understand and control immune responses.

Loss of Krox20 results in aortic valve regurgitation and impaired transcriptional activation of fibrillar collagen genes.

AIMS: Heart valve maturation is achieved by the organization of extracellular matrix (ECM) and the distribution of valvular interstitial cells. However, the factors that regulate matrix components required for valvular structure and function are unknown. Based on the discovery of its specific expression in cardiac valves, we aimed to uncover the role of Krox20 (Egr-2) during valve development and disease. METHODS AND RESULTS: Using series of mouse genetic tools, we demonstrated that loss of function of Krox20 caused significant hyperplasia of the semilunar valves, while atrioventricular valves appeared normal. This defect was associated with an increase in valvular interstitial cell number and ECM volume. Echo Doppler analysis revealed that adult mutant mice had aortic insufficiency. Defective aortic valves (AoVs) in Krox20(-/-) mice had features of human AoV disease, including excess of proteoglycan deposition and reduction of collagen fibres. Furthermore, examination of diseased human AoVs revealed decreased expression of KROX20. To identify downstream targets of Krox20, we examined expression of fibrillar collagens in the AoV leaflets at different stages in the mouse. We found significant down-regulation of Col1a1, Col1a2, and Col3a1 in the semilunar valves of Krox20 mutant mice. Utilizing in vitro and in vivo experiments, we demonstrated that Col1a1 and Col3a1 are direct targets of Krox20 activation in interstitial cells of the AoV. CONCLUSION: This study identifies a previously unknown function of Krox20 during heart valve development. These results indicate that Krox20-mediated activation of fibrillar Col1a1 and Col3a1 genes is crucial to avoid postnatal degeneration of the AoV leaflets.