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other·ophthalmology, genetics, developmental biology, molecular biology·PMC10120076

Overexpression screen of chromosome 21 genes reveals modulators of Sonic hedgehog signaling relevant to Down syndrome

Disease Models & Mechanisms · 8 authors, 9 centres

AI SUMMARY

FIDELITY 64%

POPULATIONMouse cell lines (Shh-LIGHT2, SmoA1-LIGHT, C3H10T1/2) and primary granule cell precursors from euploid mouse pups; Ts65Dn and TcMAC21 mouse models of Down syndrome

INTERVENTIONOverexpression of individual human chromosome 21 cDNAs (163 genes) via transient transfection or lentiviral transduction

COMPARISONControl cells transfected with GFP or DsRed2; euploid littermates for mouse models

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TL;DR

This study screened 163 human chromosome 21 genes and identified four (B3GALT5, ETS2, HMGN1, and MIS18A) that inhibit Sonic hedgehog (SHH) signaling and reduce proliferation of cerebellar granule cell precursors when overexpressed. These findings suggest that overexpression of specific chromosome 21 genes contributes to cerebellar hypoplasia in Down syndrome by disrupting SHH signaling. The results prioritize these genes for further mechanistic studies and potential therapeutic targeting.

Full summary

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**Background:** Trisomy 21 (Down syndrome) and mutations in the Sonic hedgehog (SHH) signaling pathway share overlapping phenotypes, including cerebellar hypoplasia, craniofacial abnormalities, and congenital heart defects. Trisomic cells from individuals with Down syndrome show deficits in SHH signaling, yet chromosome 21 does not encode any known components of the canonical SHH pathway. This study aimed to identify chromosome 21 genes that modulate SHH signaling when overexpressed, to understand their contribution to Down syndrome phenotypes.

**Methods:** The authors screened 163 human chromosome 21 cDNAs (selected for high homology to mouse genes) in multiple assays: (1) Shh-LIGHT2 and SmoA1-LIGHT mouse cell lines with a Gli1-luciferase reporter; (2) C3H10T1/2 mesenchymal stem cells measuring SHH-dependent osteoblast differentiation via alkaline phosphatase activity; (3) primary granule cell precursors (GCPs) from postnatal day 6 (P6) mouse pups, assessing proliferation via EdU incorporation after lentiviral overexpression. RNA sequencing was performed on cerebella from Ts65Dn (n=4 trisomic, 4 euploid) and TcMAC21 (n=4 trisomic, 4 euploid) mouse models at P6 to confirm expression of candidate genes. Data were integrated with previous zebrafish overexpression screens and public expression databases (BrainSpan, single-cell RNA-seq).

**Key Results:** In Shh-LIGHT2 cells treated with SAG, overexpression of 9 genes (ABCG1, CRYAA, DOP1B, DYRK1A, ITSN1, MCM3AP, N6AMT1) activated SHH signaling, while GET1 and S100B inhibited it (z-score >2 or <-2). In SmoA1-LIGHT cells, DYRK1A, IFNAR2, and MRPL39 activated, while ABCG1, KCNE1, NDUFV3, and PRMT2 inhibited signaling. Sixteen of 20 identified genes showed consistent direction of effect across both cell lines. In C3H10T1/2 cells, overexpression of ABCG1, HMGN1, JAM2, MIS18A, NDUFV3, and RWDD2B significantly reduced osteoblast differentiation (q<0.1), while CHAF1B, GET1, and PCBP3 increased it. RNA-seq showed trisomic genes were overexpressed by an average of 1.45±0.29-fold in Ts65Dn and 1.81±1.18-fold in TcMAC21 cerebella. Integration of all screens prioritized four genes—B3GALT5, ETS2, HMGN1, and MIS18A—that consistently inhibited SHH signaling and significantly reduced proliferation of primary GCPs when overexpressed (p<0.05 to p<0.0001, one-way ANOVA). For example, HMGN1 overexpression inhibited SHH in all three cell-based assays and reduced GCP proliferation.

**Clinical Implications:** This study identifies four chromosome 21 genes (B3GALT5, ETS2, HMGN1, MIS18A) as novel modulators of SHH signaling that may contribute to cerebellar hypoplasia in Down syndrome. The findings suggest that cerebellar hypoplasia results from an imbalance between inhibitory genes (e.g., HMGN1) and compensatory activators (e.g., DYRK1A). These results prioritize specific genes for mechanistic studies and potential therapeutic targeting. However, the authors caution that therapeutic interventions aimed at activating SHH (e.g., DYRK1A inhibitors) could worsen phenotypes in SHH-responsive tissues. The study also highlights the complexity of Down syndrome as a polygenic disorder, where multiple genes with small effects interact to produce phenotypes.

PICO

PPOPULATION

Mouse cell lines (Shh-LIGHT2, SmoA1-LIGHT, C3H10T1/2) and primary granule cell precursors from euploid mouse pups; Ts65Dn and TcMAC21 mouse models of Down syndrome

IINTERVENTION

Overexpression of individual human chromosome 21 cDNAs (163 genes) via transient transfection or lentiviral transduction

OOUTCOME

SHH signaling activity (luciferase reporter, alkaline phosphatase activity, EdU incorporation), gene expression (RNA-seq), cerebellar volume

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

64% · C

ORIGINAL PAPER

PMC10120076

Overexpression screen of chromosome 21 genes reveals modulators of Sonic hedgehog signaling relevant to Down syndrome

Disease Models & Mechanisms

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ABSTRACT

ABSTRACT Trisomy 21 and mutations in the Sonic hedgehog (SHH) signaling pathway cause overlapping and pleiotropic phenotypes including cerebellar hypoplasia, craniofacial abnormalities, congenital heart defects and Hirschsprung disease. Trisomic cells derived from individuals with Down syndrome possess deficits in SHH signaling, suggesting that overexpression of human chromosome 21 genes may contribute to SHH-associated phenotypes by disrupting normal SHH signaling during development. However, chromosome 21 does not encode any known components of the canonical SHH pathway. Here, we sought to identify chromosome 21 genes that modulate SHH signaling by overexpressing 163 chromosome 21 cDNAs in a series of SHH-responsive mouse cell lines. We confirmed overexpression of trisomic candidate genes using RNA sequencing in the cerebella of Ts65Dn and TcMAC21 mice, model systems for Down syndrome. Our findings indicate that some human chromosome 21 genes, including DYRK1A , upregulate SHH signaling, whereas others, such as HMGN1 , inhibit SHH signaling. Individual overexpression of four genes ( B3GALT5 , ETS2 , HMGN1 and MIS18A ) inhibits the SHH-dependent proliferation of primary granule cell precursors. Our study prioritizes dosage-sensitive chromosome 21 genes for future mechanistic studies. Identification of the genes that modulate SHH signaling may suggest new therapeutic avenues for ameliorating Down syndrome phenotypes.

CITATION EXPORT

Anna J. Moyer, Fabian-Xosé Fernandez, Yicong Li et al.. Overexpression screen of chromosome 21 genes reveals modulators of Sonic hedgehog signaling relevant to Down syndrome. Disease Models & Mechanisms. (2023). https://doi.org/10.1242/dmm.049712

AUTHORS

8 · 9 CENTRES

Anna J. Moyer, Fabian-Xosé Fernandez, Yicong Li, Donna K. Klinedinst, Liliana D. Florea, Yasuhiro Kazuki, Mitsuo Oshimura, Roger H. Reeves

1Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
2Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
3Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35233, USA
4Department of Psychology, University of Arizona, Tucson, AZ 85724, USA
5Department of Neurology, University of Arizona, Tucson, AZ 85724, USA
6BIO5 and McKnight Brain Research Institutes, Tucson, AZ 85721, USA
7Division of Genome and Cellular Functions, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine and Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
8Chromosome Engineering Research Center, Tottori University, Yonago, Tottori 683-8503, Japan
9Trans Chromosomics, Inc., 86 Nishi-cho, Yonago, Tottori 683-8503, Japan