A neuronal enhancer network upstream of MEF2C is compromised in patients with Rett-like characteristics

Mutations in myocyte enhancer factor 2C (MEF2C), an important transcription factor in neurodevelopment, are associated with a Rett-like syndrome. Structural variants (SVs) upstream of MEF2C, which do not disrupt the gene itself, have also been found in patients with a similar phenotype, suggesting that disruption of MEF2C regulatory elements can also cause a Rett-like phenotype. To characterize those elements that regulate MEF2C during neural development and that are affected by these SVs, we used genomic tools coupled with both in vitro and in vivo functional assays. Through circularized chromosome conformation capture sequencing (4C-seq) and the assay for transposase-accessible chromatin using sequencing (ATAC-seq), we revealed a complex interaction network in which the MEF2C promoter physically contacts several distal enhancers that are deleted or translocated by disease-associated SVs. A total of 16 selected candidate regulatory sequences were tested for enhancer activity in vitro, with 14 found to be functional enhancers. Further analyses of their in vivo activity in zebrafish showed that each of these enhancers has a distinct activity pattern during development, with eight enhancers displaying neuronal activity. In summary, our results disentangle a complex regulatory network governing neuronal MEF2C expression that involves multiple distal enhancers. In addition, the characterized neuronal enhancers pose as novel candidates to screen for mutations in neurodevelopmental disorders, such as Rett-like syndrome

Unraveling the transcriptional regulation of TWIST1 in limb development.

The transcription factor TWIST1 plays a vital role in mesoderm development, particularly in limb and craniofacial formation. Accordingly, haploinsufficiency of TWIST1 can cause limb and craniofacial malformations as part of Saethre-Chotzen syndrome. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Here, we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in the developing limb and branchial arches. Using available p300 and H3K27ac ChIP-seq data, we identified 12 enhancer candidates, located both within and outside the coding sequences of the neighboring gene, Histone deacetyase 9 (HDAC9). Using zebrafish and mouse enhancer assays, we showed that eight of these candidates have limb/fin and branchial arch enhancer activity that resemble Twist1 expression. Using 4C-seq, we showed that the Twist1 promoter region interacts with three enhancers (eTw-5, 6, 7) in the limb bud and branchial arch of mouse embryos at day 11.5. Furthermore, we found that two transcription factors, LMX1B and TFAP2, bind these enhancers and modulate their enhancer activity. Finally, using CRISPR/Cas9 genome editing, we showed that homozygous deletion of eTw5-7 enhancers reduced Twist1 expression in the limb bud and caused pre-axial polydactyly, a phenotype observed in Twist1+/- mice. Taken together, our findings reveal that each enhancer has a discrete activity pattern, and together comprise a spatiotemporal regulatory network of Twist1 transcription in the developing limbs/fins and branchial arches. Our study suggests that mutations in TWIST1 enhancers could lead to reduced TWIST1 expression, resulting in phenotypic outcome as seen with TWIST1 coding mutations

The MEF2C regulatory network is disrupted in patients with Rett-like characteristics

Myocyte enhancer factor 2C (MEF2C) is a core transcription factor in neurodevelopment. In the context of human disease, MEF2C mutations have primarily been associated with a severe intellectual disability syndrome that shows phenotypical overlap with Rett syndrome, a progressive neurodevelopmental disorder featured by severe mental retardation, seizures and hypotonia. Recently, several patients with a similar Rett-like phenotype have been identified that harbour structural variants (SVs) upstream of MEF2C, but not encompassing the MEF2C coding sequence itself. This suggests that alteration of regulatory interactions could play a role in this Rett-like phenotype. However, the MEF2C regulatory network is yet to be deciphered. Therefore, we dissected the MEF2C region, using Circularized Chromosome Conformation Capture (4C) sequencing, in vitro and in vivo enhancer assays. Mining literature and public variant databases, we found 11 deletions, 8 translocations and one inversion in the MEF2C regulatory region that do not directly affect the MEF2C coding sequence itself. Extensive 4C sequencing revealed an interaction network, in which the MEF2C promoter physically contacts distal, putative enhancers, located in the region affected in these patients. Using luciferase assays, we confirmed enhancer potential for 9 out of 15 selected candidate elements. Furthermore, using in vivo zebrafish enhancer assays, we showed that several elements have neuronal activity in the developing zebrafish. Five enhancers showed activity in the forebrain, three in specific neurons above the eye and one in the notochord. Interestingly, three enhancers also showed specific activity in the heart. This is not surprising, as MEF2C is also well known to be involved in heart development. In summary, we started to disentangle a complex regulatory network governing MEF2C transcription, that involves multiple distal enhancers. Disrupting this regulatory structure is likely detrimental to normal neurodevelopment and can give rise to neurodevelopmental disorders such as Rett-like syndrome

The MEF2C regulatory network is disrupted in patients with Rett-like characteristics

Myocyte enhancer factor 2C (MEF2C) is a core transcription factor in neurodevelopment. In the context of human disease, MEF2C haploinsufficiency has been associated with a Rett-like syndrome, characterized by severe intellectual disability, seizures and stereotypic movements. Chromosomal aberrations in the MEF2C region, some of which do not encompass MEF2C itself, have been associated with a similar Rett-like phenotype. So far, 11 deletions, eight translocations and one inversion involving 5q14.3 in Rett-like patients have been described, that do not directly affect the MEF2C coding sequence, suggesting that disruption of MEF2C regulatory elements could result in this Rett-like syndrome. The MEF2C regulatory network, however, is yet to be deciphered. To shed light on the regulatory interactions governing neuronal MEF2C transcription, we performed Circularized Chromosome Conformation Capture (4C) sequencing and characterized the activity of putative, neuronal MEF2C enhancers using in vitro and in vivo enhancer assays. Through extensive 4C sequencing in a neuronal cell line, we revealed an intricate interaction network, in which the MEF2C promoter physically contacts distal putative enhancers located in the region affected in Rett-like patients. We confirmed enhancer potential for 10 out of 15 selected candidate elements using luciferase assays. Moreover, eight candidate enhancers exhibited in vivo neuronal activity in zebrafish. Six of these were active in the forebrain, of which three also displayed additional activity in specific neurons above the eye and two in the notochord. Further, one enhancer displayed mid- and hindbrain activity and one was specifically active in the notochord. Interestingly, we also identified three enhancers with specific activity in the heart. This is not surprising, as MEF2C is known to be involved in heart development as well. In summary, we have begun to unravel a complex regulatory network governing neuronal MEF2C transcription, that involves multiple distal enhancers. Disrupting this regulatory structure is likely detrimental to normal neurodevelopment and can give rise to neurodevelopmental disorders such as Rett-like syndrome

A neuronal enhancer network upstream of MEF2C is compromised in patients with Rett-like characteristics

Myocyte enhancer factor 2C (MEF2C) is a core transcription factor in neurodevelopment and aberrations are typically associated with a Rett-like syndrome, featured by severe intellectual disability, seizures and stereotypic movements. However, structural variants upstream of MEF2C have been found in patients with a similar phenotype, suggesting that disruption of MEF2C regulatory elements represents the underlying genetic cause in these cases. To shed light on how these aberrations impact MEF2C regulation, we dissected the MEF2C regulatory landscape. Using Circularized Chromosome Conformation Capture (4C) sequencing in a neuronal cell line, we revealed a complex interaction network in which the MEF2C promoter physically contacts several distal enhancers located upstream of the coding sequence. Luciferase reporter assays confirmed enhancer activity for thirteen out of sixteen selected candidate enhancers. Moreover, using enhancer assays in zebrafish, we characterized eight enhancers with in vivo neuronal activity. While each of these enhancers displayed a distinct activity pattern, several showed overlapping activity in the forebrain, notochord or neurons above the eye. In summary, we disentangled a complex regulatory network governing neuronal MEF2C transcription that involves multiple distal enhancers. Disrupting this regulatory structure is likely detrimental to normal neurodevelopment and can give rise to neurodevelopmental disorders such as Rett-like syndrome

Noncoding structural variants disrupt the regulatory architecture of Rett genes

Rett syndrome is a progressive neurodevelopmental disorder, characterized by a severe developmental delay, absence of speech, seizures, hypotonia and stereotypic movements. It is typically caused by mutations in the MECP2 gene, but several other genes, including the transcription factors MEF2C and FOXG1, have been associated with a Rett-like phenotype as well. Recently, we and others identified several noncoding structural variants (SVs) in patients with Rett-like characteristics. All SVs are located proximal to the coding sequence of MEF2C or FOXG1, suggesting disruption of the regulatory structure governing these genes. Using Circularized Chromosome Conformation Capture (4C) sequencing in a neuronal cell line, we identified a complex regulatory interaction network in the MEF2C region. We found that the MEF2C promoter physically contacts multiple distal enhancer regions upstream of its coding sequence. Based on epigenetic enhancer marks and sequence conservation, we delineated 16 putative enhancer elements, of which 14 were active in in vitro luciferase assays and 8 displayed in vivo neuronal activity during zebrafish development. For FOXG1 as well, 4C-seq experiments have shown that the promoter interacts with at least three in vivo validated brain enhancers, all situated in a region affected by deletions or translocations in multiple Rett-like patients. In summary, Rett genes MEF2C and FOXG1 are part of complex regulatory networks involving multiple distal enhancers. Disruption of these regulatory structures by noncoding SVs could form the genetic basis of the Rett-like phenotype observed in some patients