Abstract
Background FOXG1 has important functions for neuronal differentiation and balances excitatory/inhibitory network activity. Mutations in the human FOXG1 gene cause a rare neurodevelopmental disorder, FOXG1-syndrome, which manifests differing phenotypes, including severe cognitive dysfunction, microencephaly, social withdrawal, and communication and memory deficits. Changes at the molecular level underlying these functional abnormalities upon FOXG1 haploinsufficiency are largely unexplored, in human patients as well as in animals modelling the debilitating disease.
Methods We present multi-omics data and explore comprehensively how FOXG1 impacts neuronal maturation at the chromatin level in the adult mouse hippocampus. We used RNA-, ATAC- and ChIP-sequencing of primary hippocampal neurons and co-immunoprecipitation to explore various levels of epigenetic changes and transcription factor networks acting to alter neuronal differentiation upon reduction of FOXG1.
Results We provide the first comprehensive multi-omics data set exploring FOXG1 presence at the chromatin and identifying the consequences of reduced FOXG1 expression in primary hippocampal neurons. Analyzing the multi-omics data, our study reveals that FOXG1 uses various different ways to regulate transcription at the chromatin level. On a genome-wide level, FOXG1 (i) both represses and activates transcription, (ii) binds mainly to enhancer regions, and (iii) bidirectionally alters the epigenetic landscape in regard to levels of H3K27ac, H3K4me3, and chromatin accessibility. Genes affected by the chromatin alterations upon FOXG1 reduction impact synaptogenesis and axonogenesis. This finding emphasizes the importance of FOXG1 to integrate and coordinate transcription of genes necessary for proper neuronal function by acting on a genome-wide level. Interestingly, FOXG1 acts through histone deacetylases (HDACs) and inhibition of HDACs partly rescued transcriptional alterations observed upon FOXG1 reduction. On a more detailed level of analysis, we show that FOXG1 (iv) operates synergistically with NEUROD1. Interestingly, we could not detect a clear hierarchy of these two key transcription factors, but instead provide first evidence that they act in highly concerted and orchestrated manner to control neuronal differentiation.
Conclusions This integrative and multi-omics view of changes upon FOXG1 reduction reveals an unprecedented multimodality of FOXG1 functions converging on neuronal maturation, fueling novel therapeutic options based on epigenetic drugs to alleviate, at least in part, neuronal dysfunctions.
Competing Interest Statement
The authors have declared no competing interest.
List of abbreviations
- ATAC
- Assay for Transposase-Accessible Chromatin
- BSA
- Bovine serum albumin
- CA
- Cornu ammonis
- CNS
- Central nervous system
- DB
- Database
- DEG
- Differentially expressed gene
- DG
- Dentate gyrus
- DIV
- Days in vitro
- DMEM
- Dulbecco’s Modified Eagle’s Mediu
- DMSO
- Dimethyl sulfoxide
- DPBS
- Dulbecco’s phosphate buffered saline
- EDTA
- Ethylenediaminetetraacetic acid
- FBS
- Fetal bovine serum
- FGF
- Fibroblast growth factor
- FKH
- Forkhead
- FOXA1
- Forkhead box A 1
- FOXG1
- Forkhead box G 1
- FOXO
- Forkhead box O
- GABA
- Gamma-Aminobutyric acid
- GAPDH
- Glyceraldehyde-3-phosphate dehydrogenase
- GFP
- Green fluorescent protein
- H3K27
- Histone 3 lysine 27
- H3K4
- Histone 3 lysine 4
- H3K79
- Histone 3 lysine 79
- HAT
- Histone acetyl transferase
- HBSS
- Hank’s buffered salt solution
- HDAC
- Histone deacetylase
- HEPES
- N-2-hydroxyethylpiperazine-n’-2-ethanesulfonic acid
- IP
- Immunoprecipitation
- ISH
- In stu hybridization
- ISL1
- Isl lim homeobox 1
- KD
- Knockdown
- KI
- Knockin
- LFC
- Log fold change
- MECP2
- Methyl cpg binding protein 2
- N2A
- Neuro2a neuroblastoma
- NEAA
- Non-essential amino acids
- NEUROD1
- Neurogenic differentiation 1
- NTP
- Nucleoside triphosphate
- OE
- Overexpression
- PBS
- Phosphate buffered saline
- PFA
- Paraformaldehyde
- PIC
- Proteinase inhibitor cocktail
- PMSF
- Phenylmethylsulfonyl fluoride
- PSN
- Penicillin-streptomycin-neomycin
- QC
- Quality control
- RELACS
- Restriction enzyme-based labeling of chromatin in situ
- ROR
- Retinoic acid-related Orphan Receptors
- RPKM
- Reads per kilo base per million mapped reads
- RT
- Reverse transcription
- SATB2
- Special AT-rich sequence-binding protein 2
- SDS
- Sodium dodecyl sulfate
- SEM
- Standard error of mean
- SEMA5A
- Semaphorin 5A
- SIRT1
- Sirtuin 1
- SNP
- Single nucleotide polymorphism
- SOX2
- Sry-box transcription factor 2
- TBS
- Tris buffered saline
- TBST
- Tris buffered saline with tween
- TF
- Trasncription factor
- TGF
- Transforming growth factor
- TPM
- Transcript per million
- TRAP
- Transcription factor Affinity Prediction
- TSA
- Trichostatin A
- TSS
- Transcription start site
- UMI
- Unique molecular identifiers
- WT
- Wild type
- ZBTB18
- Zinc finger and btb domain containing 18
- ZBTB20
- Zinc finger and btb domain containing 20