Transcriptional regulation of SVZ adult neurogenesis by TALE-homeodomain protein PBX1

Abstract

Neurogenesis begins in early embryonic development and continues lifelong in neurogenic niches of the adult mammalian brain. One of these niches is the subventricular zone (SVZ), lining the lateral ventricles of the adult brain. Differentiation processes such as neurogenesis are coordinated by the protein group of transcription factors (TFs), which bind to DNA marked by a specific nucleotide composition referred to as ’motifs’ and recruit effector proteins. One transcription factor critical for neurogenesis is the three amino acid loop extension-homeodomain (TALE-HD) protein pre-B cell leukemia homeobox 1 (PBX1). While previous studies had explored the impact of PBX1 on embryonic cortical development, and a role in adult SVZ neurogenesis was demonstrated, thus far no genome-wide assessment of transcriptional modulation by PBX1 was performed in the adult context. Therefore, as transcription factors do not act in solicitude, but in concert with an abundance of interactors in gene regulatory networks (GRNs), the PBX1 interactome active in this context was assessed by a coordinated program of genome-wide approaches. As model system, primary neural stem- and progenitor cells derived from the murine SVZ, cultivated as adult neurospheres (aNS), were utilized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) uncovered PBX1 binding to a large number of different genomic sites in aNS cells, implicating PBX1 in neurogenic differentiation as well as broader biological functions. While prominent PBX1-enriched sites, termed ’peaks’, were largely associated with promotor regions, smaller peaks were preferentially localized at intronic and distal intergenic sites. Further, PBX1 peaks preferentially co-localized with activating as opposed to repressing histone modifications. ATAC-seq in aNS cells followed by DNA footprinting supported the notion of PBX1 binding in open chromatin, associated with gene activity. Subsequently, PBX1 ChIP-seq data were subjected to motif analysis and integrated with motif enrichment of open, accessible chromatin regions to predict interaction partners. Potential interactors were then substantiated by further bioinformatic analysis, including intersection of aNS PBX1 ChIP-seq peaks with neurodevelopmental datasets of interaction candidates derived from the public domain. Additionally, GRN analysis of PBX1 ChIP-seq and genes affected by Pbx1 kd as assessed by RNA-seq analysis complemented this prediction. Finally, mass spectrometry (MS) analysis of PBX1-immunoprecipitation was performed in aNS cells, validating association with several of the predicted interactors, including SOX2, OLIG2, and NFI transcription factors. Importantly, this approach further confirmed TCF4 and TCF12, both members of the bHLH class I transcription factor family (TCFs) as PBX1 interactors. These two transcription factors, together with the closely related TCF3, had formerly been described as modulators of neurogenesis, in both adult SVZ and embryonic contexts. While genomic alteration produces a TCF3-PBX1 fusion proteins in an aggressive subtype of pre-B cell leukemia by t(1;19) translocation, and an interaction between PBX1 and TCF3 proteins has been suggested in vitro, physiological interactions of PBX1 with TCF factors to date have not been described. Within this thesis, co-localization of PBX1 and TCF3 had been predicted in silico but no interaction was observed in MS. Thus, co-immunoprecipitation and proximity ligation assay were employed to confirm complex formation between both factors. Functional interaction between PBX1 and TCF3 or TCF4 was tested by siRNA mediated knock-down (kd) of either factor followed by RNA-seq, revealing common target genes. Assessment of neurodevelopmental PBX1 and TCF3 intersecting ChIP-seq peaks revealed binding motifs for both factors in close proximity, suggesting direct interaction on chromatin. Further, visualizing ChIP-seq profiles at example loci such as Olig2 revealed shared binding patterns for PBX1 and TCF3/4. Interestingly, several genes involved in DNA replication were commonly downregulated upon depletion of Pbx1 or Tcf3, while genes implicated in neuronal differentiation were upregulated upon either kd. Despite this observation, depletion of Pbx1, Tcf3 or Tcf4 in aNS cells followed by in vitro differentiation led to diminished neuronal fate commitment. Together, this suggests a role for PBX1-TCF interactions in balancing progenitor cell proliferation with neur(on)al differentiation. Collectively, these results suggest a previously unrecognized cooperation of PBX1 and class I bHLH transcription factors in coordinating neural progenitor proliferation and neuronal differentiation during adult forebrain neurogenesis. This novel link further raises the possibility that the leukemogenic fusion protein TCF3-PBX1 hijacks the physiological cooperation of both transcription factors and adapts it to a pathological context.