Expression Profiling Identifies TWIST2 Target Genes in Setleis Syndrome Patient Fibroblast and Lymphoblast Cells

Abstract

Background: Setleis syndrome (SS) is a focal facial dermal dysplasia presenting with bilateral temporal skin lesions, eyelash abnormalities and absent meibomian glands. SS is a rare autosomal recessive disorder caused by mutations in the TWIST2 gene, which codes for a transcription factor of the bHLH family known to be involved in skin and facial development. Methods: We obtained gene expression profiles by microarray analyses from control and SS patient primary skin fibroblast and lymphoblastoid cell lines. Results: Out of 983 differentially regulated genes in fibroblasts (fold change ≥ 2.0), 479 were down-regulated and 509 were up-regulated, while in lymphoblasts, 1248 genes were down-regulated and 73 up-regulated. RT-PCR reactions confirmed altered expression of selected genes. Conclusions: TWIST2 is described as a repressor, but expression profiling suggests an important role in gene activation as well, as evidenced by the number of genes that are down-regulated, with a much higher proportion of down-regulated genes found in lymphoblastoid cells from an SS patient. As expected, both types of cell types showed dysregulation of cytokine genes. These results identify potential TWIST2 target genes in two important cell types relevant to rare disorders caused by mutations in this bHLH gene.

Mechanisms of Regulation of the CHRDL1 Gene by the TWIST2 and ADD1/SREBP1c Transcription Factors

“…We then assessed the effect of TWIST1, TWIST2 and the TWIST2-Q119X mutant when co-expressed with SREBP1c. As seen in Figure 11 B, SREBP1c activation of the reporter gene was significantly blocked by TWIST2, but not by TWIST1 or TWIST2-Q119X, which may explain why CHRDL1 gene expression is elevated in patient dermal fibroblasts [ 29 ]. Whether that requires binding by TWIST2 homodimers to the upstream CHRDL1 gene region we tested by EMSAs (probe #5), which the homodimers of the Q119X mutant protein failed to do, or formation of heterotrimeric complexes that may or may not bind DNA, deserves further study.…”

“…As already mentioned in the introduction, the CHRDL1 gene is a differentially up-regulated gene in Setleis syndrome patient dermal fibroblast cells of [ 29 ]. Hence, we hypothesized that TWIST2 acts as a repressor in CHRDL1 gene expression, since the truncated TWIST2-Q119X mutant protein expressed in patients appears to lack the capacity to repress its expression.…”

“…The Q119X mutant form of TWIST2 forms homo- and heterodimers with E12 and TWIST1, and binds E-boxes in the human Periostin ( POSTN ) gene promoter [ 28 ]. Expression profiling of RNAs derived from Puerto Rican Setleis syndrome patient dermal fibroblasts revealed a long list of differentially regulated genes, from which the chordin-like 1 ( CHRDL1 ) gene stands out as having the highest positive fold-change, being up-regulated 74-fold [ 29 ]. This finding suggests that the truncated Q119X TWIST2 protein is a negative regulator of CHRDL1 gene expression.…”

Single-Cell and Bulk Transcriptome Data Integration Reveals Dysfunctional Cell Types and Aberrantly Expressed Genes in Hypertrophic Scar

“…Consistently, CRBE3L1 has been identified as one of the critical TFs involved in fibrotic skin diseases by a scRNA-seq study ( Deng et al, 2021 ). The other TF, TWIST2, was relevant to Setleis syndrome (SS), a focal facial dermal dysplasia presenting with bilateral temporal skin lesions ( Crespo et al, 2021 ). Moreover, TWIST1, an important paralog of TWIST2, was involved in the fibrogenesis of keloid fibroblasts, and might serve as a therapeutic target of keloid ( Liu et al, 2021 ), suggesting that TWIST2 might also be considered as a promising therapeutic target in HS.…”

“…Also, activation of PI3K/AKT pathway could lead to hypertrophic scar formation and ECM deposition via upregulated expression of Collagen I, Collagen III, α-SMA, and Cleaved caspase-3 in hypertrophic scar fibroblasts ( Xiao, 2020 ; Zhi et al, 2021 ). In addition, some transcription factors (TFs) such as CRBE3L1 and TWIST2 have been identified as critical regulators in skin diseases ( Crespo et al, 2021 ; Deng et al, 2021 ). Importantly, the two TFs were also involved in kidney fibrosis ( Grunz-Borgmann et al, 2017 ; Yamamoto et al, 2021 ).…”