Non-coding/epigenetic regulation

PROJECT 3 - SUMMARY
Adolescent idiopathic scoliosis (AIS) affects ~3% of the population worldwide and is estimated to cost several
billion dollars annually in surgeries alone in the US. The causes of AIS remain largely unknown. While
mutations in genes leading to syndromic scoliosis (associated with other symptoms) have been discovered, the
identification of mutations causing non-syndromic/isolated AIS (only AIS without any other symptoms), have
been less successful. Several genome-wide association studies (GWAS) have identified AIS-associated single
nucleotide polymorphisms (SNPs) in noncoding regions adjacent to promising candidate genes, suggesting a
role for gene regulatory sequences, such as enhancers, in AIS. In our preliminary results, we show that the
knockout of a spinal cord enhancer near an AIS GWAS associated region residing in the PAX1 locus, a gene
known to be involved in spinal development, is associated with a kinky tail phenotype, similar to the Pax1 gene
knockout and hypomorphic mutation. Interestingly, the kinky tail phenotype is more apparent in females, fitting
with the female specific AIS GWAS association that we observed for this region. Another hurdle in AIS
genetics is that until recently there were no specific tissue/s whose aberration was widely known to cause AIS.
Through work carried out by all three of our projects along with other investigators, the the cartilage
extracellular matrix (ECM), i.e. matrisome, was found to be strongly implicated in the pathogenesis of AIS.
Here, we will use total RNA-seq to characterize the noncoding RNAs expressed in these tissues. In addition,
using combined single-cell RNA/ATAC-seq on the cartilage matrisome to identify the genes and regulatory
elements associated with AIS in a single cell manner. In addition, we will carry out H3K4me3 (a mark for active
promoters) and H3K27ac (a mark for active promoters and enhancers) Hi-ChIP, a technique that uses
chromatin conformation in combination with chromatin immunoprecipitation (ChIP) to capture specific
chromosomal interactions, to identify the target genes of these AIS-associated regulatory elements. Our
genomic datasets will also feed into Project 1 (Human) and Project 2 (Zebrafish) providing candidate genes
and regulatory elements to screen for AIS-associated mutations/phenotypes. To functionally characterize AIS-
associated regulatory elements, we will use regulatory element assays in cell lines and mice combined with
mouse knockouts to characterize gene regulatory sequences that are associated with AIS. These sequences
will be selected from GWAS, both from the literature and Project 1 (Human), whole-genome sequencing on
individuals with AIS carried out by Project 1 (Human) and near genes shown to cause AIS in zebrafish from our
Project 2 (Zebrafish). Our preliminary results for the Pax1 enhancer knockout already attest for the potential of
this approach. Combined, our work will provide a genomic encyclopedia of genes and regulatory elements that
could be associated with AIS and serve as a model for the functional characterization of gene regulatory
elements involved in additional subtypes of scoliosis, musculoskeletal and other human disease.

Grant Number: 5P01HD084387-09
NIH Institute/Center: NIH
Principal Investigator: Nadav Ahituv