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.

Functional characterization of the ZEB2 regulatory landscape.

Zinc finger E-box-binding homeobox 2 (ZEB2) is a key developmental regulator of the central nervous system (CNS). Although the transcriptional regulation of ZEB2 is essential for CNS development, the elements that regulate ZEB2 expression have yet to be identified. Here, we identified a proximal regulatory region of ZEB2 and characterized transcriptional enhancers during neuronal development. Using chromatin immunoprecipitation sequencing for active (H3K27ac) and repressed (H3K27me3) chromatin regions in human neuronal progenitors, combined with an in vivo zebrafish enhancer assay, we functionally characterized 18 candidate enhancers in the ZEB2 locus. Eight enhancers drove expression patterns that were specific to distinct mid/hindbrain regions (ZEB2#e3 and 5), trigeminal-like ganglia (ZEB2#e6 and 7), notochord (ZEB2#e2, 4 and 12) and whole brain (ZEB2#e14). We further dissected the minimal sequences that drive enhancer-specific activity in the mid/hindbrain and notochord. Using a reporter assay in human cells, we showed an increased activity of the minimal notochord enhancer ZEB2#e2 in response to AP-1 and DLX1/2 expressions, while repressed activity of this enhancer was seen in response to ZEB2 and TFAP2 expressions. We showed that Dlx1 but not Zeb2 and Tfap2 occupies Zeb2#e2 enhancer sequence in the mouse notochord at embryonic day 11.5. Using CRISPR/Cas9 genome editing, we deleted the ZEB2#e2 region, leading to reduction of ZEB2 expression in human cells. We thus characterized distal transcriptional enhancers and trans-acting elements that govern regulation of ZEB2 expression during neuronal development. These findings pave the path toward future analysis of the role of ZEB2 regulatory elements in neurodevelopmental disorders, such as Mowat-Wilson 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.

Mutations in the microtubule-associated protein MAP11 (C7orf43) cause microcephaly in humans and zebrafish.

Microtubule associated protein 11 (MAP11, previously termed C7orf43) encodes a highly conserved protein whose function is unknown. Through genome-wide linkage analysis combined with whole exome sequencing, we demonstrate that human autosomal recessive primary microcephaly is caused by a truncating mutation in MAP11. Moreover, homozygous MAP11-orthologue CRISPR/Cas9 knock-out zebrafish presented with microcephaly and decreased neuronal proliferation, recapitulating the human phenotype. We demonstrate that MAP11 is ubiquitously transcribed with high levels in brain and cerebellum. Immunofluorescence and co-immunoprecipitation studies in SH-SY5Y cells showed that MAP11 associates with mitotic spindles, co-localizing and physically associating with α-tubulin during mitosis. MAP11 expression precedes α-tubulin in gap formation of cell abscission at the midbody and is co-localized with PLK1, a key regulator of cytokinesis, at the edges of microtubule extensions of daughter cells post cytokinesis abscission, implicating a role in mitotic spindle dynamics and in regulation of cell abscission during cytokinesis. Finally, lentiviral-mediated silencing of MAP11 diminished SH-SY5Y cell viability, reducing proliferation rather than affecting apoptosis. Thus, MAP11 encodes a microtubule-associated protein that plays a role in spindle dynamics and cell division, in which mutations cause microcephaly in humans and zebrafish.