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

Association analysis identifies ZNF750 regulatory variants in psoriasis

<p>Abstract</p> <p>Background</p> <p>Mutations in the <it>ZNF750 </it>promoter and coding regions have been previously associated with Mendelian forms of psoriasis and psoriasiform dermatitis. <it>ZNF750 </it>encodes a putative zinc finger transcription factor that is highly expressed in keratinocytes and represents a candidate psoriasis gene.</p> <p>Methods</p> <p>We examined whether <it>ZNF750 </it>variants were associated with psoriasis in a large case-control population. We sequenced the promoter and exon regions of <it>ZNF750 </it>in 716 Caucasian psoriasis cases and 397 Caucasian controls.</p> <p>Results</p> <p>We identified a total of 47 variants, including 38 rare variants of which 35 were novel. Association testing identified two <it>ZNF750 </it>haplotypes associated with psoriasis (p < 0.05). We also identified an excess of rare promoter and 5'untranslated region (UTR) variants in psoriasis cases compared to controls (p = 0.041), whereas there was no significant difference in the number of rare coding and rare 3' UTR variants. Using a promoter functional assay in stimulated human primary keratinocytes, we showed that four <it>ZNF750 </it>promoter and 5' UTR variants displayed a 35-55% reduction of <it>ZNF750 </it>promoter activity, consistent with the promoter activity reduction seen in a Mendelian psoriasis family with a <it>ZNF750 </it>promoter variant. However, the rare promoter and 5' UTR variants identified in this study did not strictly segregate with the psoriasis phenotype within families.</p> <p>Conclusions</p> <p>Two haplotypes of <it>ZNF750 </it>and rare 5' regulatory variants of <it>ZNF750 </it>were found to be associated with psoriasis. These rare 5' regulatory variants, though not causal, might serve as a genetic modifier of psoriasis.</p

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph )

ZNF750 is expressed in differentiated keratinocytes and regulates epidermal late differentiation genes.

Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype reminiscent of psoriasis and seborrheic dermatitis. Here we show that ZNF750 is a nuclear protein bearing a functional C-terminal nuclear localization signal. ZNF750 was specifically expressed in the epidermal suprabasal layers and its expression was augmented during differentiation, both in human skin and in-vitro, peaking in the granular layer. Silencing of ZNF750 in Ca2+-induced HaCaT keratinocytes led to morphologically apparent arrest in the progression of late differentiation, as well as diminished apoptosis and sustained proliferation. ZNF750 knockdown cells presented with markedly reduced expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, overexpression of ZNF750 in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation and with its downstream targets can serve in future elucidation of therapeutics for common diseases of skin barrier

<i>ZNF750</i> silencing in HaCaT keratinocytes.

<p>HaCaT cells were transduced with scrambled shRNA (control) or with three different ZNF750 shRNAs (shRNA-a, b, and c). Cells were harvested and assayed at day 12 of Ca²⁺ induction. (A) QRT-PCR of <i>ZNF750</i> mRNA expression in the stable transduced cell lines. Error bars represent mean values±SD, N = 3. (B) western blot analysis showing ZNF750 protein levels in the stable transduced cell lines. A total of 50 µg of protein was loaded in each sample. Actin levels were measured to ensure equal amounts of loaded protein were loaded. (C) Morphological studies at different time points in HaCaT cell differentiation: control vs. <i>ZNF750</i> shRNA-a transduced cultures examined by phase contrast microscopy during Ca²⁺ induction. (D,E) <i>ZNF750</i> downregulation enhances cell proliferation. (D) Ki67 staining (green) followed by confocal microscopy. To-Pro 3 nuclear staining is shown in blue (E) quantification of Ki67 positive cells. More than 800 cells were counted for each slide, Error bars represent mean values±SD, N = 6 (***:P<0.0001). (F,G) Effects of <i>ZNF750</i> downregulation on apoptosis as assessed by Annexin V apoptosis assay. (F) Annexin V apoptosis assay using flow cytometry analysis. (G) quantification of Annexin V positive cells. More than 50,000 cells were counted in each sample Error bars represent mean values±SD, N = 4, (***:P<0.0001). (H) Effects on cell size (X axis) and granularity (Y axis) measured by flow cytometry.</p

<i>ZNF750</i> mRNA and protein expression in Ca²⁺ induction and in human skin.

<p>(A) QRT-PCR of <i>ZNF750</i> mRNA expression during Ca²⁺ induction in NHEK cells. <i>Filaggrin</i> mRNA levels serve as internal control. Error bars represent mean values±SD, N = 3. (B) QRT-PCR of <i>ZNF750</i> mRNA expression during Ca²⁺ induction in HaCaT cells. Error bars represent mean values±SD, N = 3. (C) ZNF750 protein levels during Ca²⁺ induction in HaCaT cells, analyzed by anti-ZNF750 antibodies against both N-terminal and C-terminal regions. (D) QRT-PCR of <i>ZNF750</i>, <i>KRT1</i> and <i>FLG</i> mRNA expression in NHEK cells that were treated with PMA. Error bars represent mean values±SD, N = 2.</p

ZNF750 subcellular localization and functional NLS characterization.

<p>(A) Western blot analysis using cytoplasmic (C) and nuclear (N) fractions from HaCaT cells at proliferative/undifferentiated (0.06 mM Ca²⁺) or differentiated (1.8 mM Ca²⁺) state, analyzed by anti-ZNF750 Ab. (B) Confocal subcellular localization of ZNF750 in HaCaT cells. Cells at proliferative and differentiated states were fixed and immunostained by anti-ZNF750 Ab (red). DAPI (blue) was used to identify nuclei. Scale bar  = 20 µm. (C) Sequence alignments representing the three putative NLSs of ZNF750 homologues in various organisms. Conserved amino acids of the predicted NLSs are shaded. (D) Schematic representation of EGFP-ZNF750 constructs. (E) Confocal subcellular localization of ZNF750 constructs in HEK293 cells. The expression of pEGFP-C2 (EGFP alone) is shown as a control. EGFP (green); DAPI (blue) was used to identify nuclei. Scale bar = 20 µm.</p