Understanding the function of AHDC1 using in vitro models

Xia-Gibbs syndrome (XIGIS; OMIM #615829) is a rare neurodevelopmental disorder caused by de novo heterozygous mutations in the AT-Hook DNA-Binding Motif-Containing 1 (AHDC1) gene (OMIM# 615790). XIGIS is a phenotypically heterogeneous disorder in which patients usually present severe developmental delays with symptoms of autism spectrum disorders. The syndrome was genetically characterised in 2014 and, since then, more than 270 cases have been reported. Human AHDC1 is a protein-coding gene with a single 4.9-kb coding exon, which encodes a potentially intrinsically disordered protein of 1,603 amino acids, containing two AT-hook DNA binding motifs and a PDZ binding domain consensus sequence. All identified XIGIS-associated mutations are found in the single coding exon and likely lead to the translation of AHDC1 truncated forms, which could be involved in defective neural development, causing the neurological features of XIGIS. Recently, using different cellular models, the binding of AHDC1 to the DNA and its role in DNA methylation have been proposed, as well as its involvement in gene expression regulation, acting as a regulatory hub between enhancers and promoters to maintain gene-specific chromatin contacts. Despite this, much remains unknown about AHDC1 functions. Thus, our research focuses on further characterising AHDC1 role in cellular processes. Since the presence of AT-hook DNA binding motifs and its nuclear localisation suggest that AHDC1 might bind DNA, we investigated its possible role in gene expression regulation by identifying genes responding to AHDC1 perturbation using in vitro cellular models (SH-SY5Y and U-87MG cells, of neuronal and glial origin, respectively). Through RNA-sequencing analysis upon AHDC1 silencing, we found that differentially expressed genes are enriched for developmental process regulation pathways. Then, to better understand AHDC1 protein functions, we searched for its possible interactors by screening large-scale protein-protein interaction databases, which have already reported that AHDC1 interacts with several nuclear proteins. By coupling AHDC1 overexpression with co-localisation and co-immunoprecipitation analyses, we validated that AHDC1 binds to HP1α and HP1β within the nuclei. Additionally, HP1 ChIP-sequencing experiments following AHDC1 silencing suggest an increased HP1 binding in the absence of AHDC1, supporting the notion that AHDC1 may play a role in influencing chromatin organisation and transcriptional outcomes. Further analyses will be crucial to better define the interplay between AHDC1 and HP1 partners. Lastly, we also employed patient-derived lymphoblastoid cell lines to explore the impact of AHDC1 mutations on cellular functions. Preliminary results suggested that AHDC1 is expressed at both RNA and protein levels, with variations that may be attributed to individual variability. Although additional analysis will be needed to fully understand the consequences of the mutations, the presence of possible mutant mRNAs might lead to the production of truncated forms of the AHDC1 protein, which could have implications for its normal cellular functions. In conclusion, our findings suggest that AHDC1 may play a crucial role in regulating developmental processes, possibly through its effects on gene expression and chromatin structure in cooperation with HP1 proteins. Further investigations could deepen the specific molecular mechanisms underlying these observations and their functional implications, possibly helping to identify the mechanisms at the basis of XIGIS.