Abstract 2026 Targeting transcription factors dominantly misregulated by developmental linker histone mutant

Chromatin, a complex of DNA and proteins, is the physiological substrate of all genetic processes in eukaryotic cells. Histone proteins, encoded by replication-dependent and -independent gene families, represent a major constituent of chromatin, and their post-translational modifications have long been recognized among the major regulators of gene expression in all eukaryotic cells. Both somatic and germline mutations in the proteins involved in establishment ("writers" and "erasers") and interpretation ("readers") of histone modifications frequently arise in many human diseases, including malignancies and developmental disorders. More recently, histone genes themselves were implicated in several neurodevelopmental phenotypes, including intellectual disability (ID) and autism spectrum disorders (ASD). Yet, despite significant progress, understanding the molecular mechanisms underlying these diseases is largely lacking. A heterozygous germline mutation in linker histone H1.4 was identified in many unrelated patients with childhood overgrowth, ID and ASD. All H1.4 mutations are short indels that result in a stereotypical frameshift that results in replacement of the wild-type, lysine-rich C-terminal tail of H1.4 with a glutamic acid-rich de novo peptide, remarkably identical in all patients. Despite the dramatic change in charge, the levels of H1.4 remain unchanged, and the mutant resembles the wild-type linker histone in biochemical assays. However, our RNA-Seq and ATAC-Seq analysis identified aberrant activation of neurodevelopmental programs that were coincident with focal increase of accessibility at a subset of approximately 2000 genomic sites associated with a class of basic leucine zipper (bZIP) transcription factors. Seven paralogous bZIP transcription factors from C/EBP and sMAF families were identified as ectopic interactors of H1.4 mutants in pulldown and mass-spectrometry assays. Genomic (CUT&RUN) and imaging data (proximity ligation assays and fluorescence recovery after photobleaching) demonstrate that accumulation of mutant H1.4 drives aberrant retention of bZIP factors in chromatin. As such, these present a potential target to alleviate the cellular phenotypes driven by H1.4 mutation. Taking advantage of endogenous tRNA processing machinery, we designed a sgRNA-tRNA scaffold to express multiple guide RNAs from a single polycistronic transcript, simultaneously targeting all bZIP factors implicated in ectopic H1.4 interaction. Experiments are underway to test a genetic rescue system using an inducible CRISPRi system. These studies will test whether a developmental histone mutation can be rescued by manipulating a downstream transcription factor target.