Identification of HMX1 target genes: a predictive promoter model approach.
OBJECTIVE
A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes.


METHODS
We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently.


RESULTS
The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay.


CONCLUSIONS
Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.
Institute for Research in Ophthalmology, Sion, Switzerland.
Arnaud
A
Boulling
Linda
L
Wicht
Daniel F
DF
Schorderet
D000818:Animals; D001483:Base Sequence; D019295:Computational Biology / Q000379:methods*; D057075:Enzyme Assays; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005820:Genetic Testing; D016678:Genome / Q000235:genetics; D050487:Glycine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008957:Models, Genetic; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D049031:Sarcoglycans / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D050487:Glycine Plasma Membrane Transport Proteins; D018398:Homeodomain Proteins; C529125:NKX5-3 protein, mouse; D012097:Repressor Proteins; D049031:Sarcoglycans; C493267:Slc6a9 protein, mouse; D014157:Transcription Factors; C489362:Tshz2 protein, mouse; D008156:Luciferases