Cellular and molecular function of Uhrf1 in neural stem cells

Central Nervous System (CNS) injuries and neurodegenerative diseases can lead to a critical loss of neurons and other nerve cells, which often in mammals cannot be regenerated by the organism itself. To recover the neural damage, great efforts are being undertaken in the field of neuronal reprogramming and other modes of repair. In order to execute neural repair, a thorough understanding of the underlying molecular mechanisms of neurogenesis is required. Although great strides have been made in uncovering key transcription factors and other modulators of neurogenesis, the emerging field of neuroepigenetics is still in its infancy. Many studies have individually highlighted the importance of novel and key players of embryonic and adult neurogenesis. However, less is known about similarities and differences between the molecular mechanisms underlying neurogenesis at these two stages. To shed light on these processes, we decided to employ a candidate approach, focusing on a factor which is expressed highly in both embryonic and adult neural stem cells and progenitors and downregulated in differentiated cells. Our rationale was that such a factor could play a key role in instructing neurogenesis and allow a comparison of embryonic and adult neurogenesis. To identify new factors playing roles in neurogenesis, we mined data from transcriptional profiling carried out by our lab previously. Our lab designed protocols to isolate embryonic progenitors from the developing cerebral cortex (Pinto et al. 2008) and adult neural stem cells (NSCs) and their progeny from the subependymal zone (SEZ) (Beckervordersandforth et al. 2010; Fischer et al. 2011). From these data, we screened for epigenetic regulators of neurogenesis and identified Uhrf1 (Ubiquitin-like with PHD and RING Finger Domain 1). Uhrf1 is a multi-domain protein, described to play a role in DNA methylation, histone methylation and ubiquitination. Previous data from our lab indicated that Uhrf1 is highly expressed in the embryonic cortex by progenitors and adult neural stem cells in development and adulthood and played an important role in neurogenesis (Bayam. 2014). We observed that loss of Uhrf1 in the developing cortex primarily resulted in impaired terminal neuronal differentiation, culminating in severe postnatal death. Surprisingly, we did not observe significant changes in cell fate, progenitor proliferation or initial neuronal differentiation. Analysis of transcriptional changes largely mirrored the cellular phenotypes observed. Additionally, we could identify transcriptional programs related to cellular stress to be activated in the cortical conditional knockout (cKO) of Uhrf1. The most striking effects we observed were in the activation of specific retroviral elements (type of transposable element) known as IAPs in the cKO, which are normally silenced in control animals. We determined a loss of DNA methylation of several loci, including several groups of transposable elements. Interestingly, we could detect a selective increase of 5-hydroxy methyl cytosine (5hmC, a mark associated with transcriptional activation) on Intracisternal A-Particles (IAPs), as opposed to other elements. We could perform molecular rescue experiments to elucidate interplay between Uhrf1 and Tet enzymes in the regulation of IAP elements, in cortical neural stem cells. To examine the role of Uhrf1 in adult neurogenesis we used the GlastcreERT2 mice (Mori et al. 2006) and CAG reporter mice (Nakamura et al. 2006) to inducibly delete Uhrf1 in adult neural stem cells and all their progeny. As opposed to the role of Uhrf1 in embryonic neurogenesis, we observed strong changes in progenitor proliferation in the adult cKO of Uhrf1 in the SEZ. Moreover, in contrast to embryonic stages, we could not detect significantly altered cell death in the adult cKO. Analysis of the molecular role of Uhrf1 in this niches, uncovered a surprising difference in IAP regulation between the SEZ and dentate gyrus. Although we detected upregulation of IAP structural proteins in the SEZ cKO, this was not the case in the dentate gyrus cKO. However, the dentate gyrus adult neurogenesis was severely impaired, with much reduced proliferation and neuroblasts. Thus, the cellular and molecular function of Uhrf1 is clearly different in adult neurogenesis compared to development. The data from my thesis allowed us to draw several key conclusions regarding the molecular role of Uhrf1. Firstly, we observed specificity in regulation of retroviral elements by Uhrf1 in the embryonic cortex and adult SEZ. Secondly, we uncovered a role for factors expressed in early neural stem cells (in this case, Uhrf1) to exert long-term effects on their progeny. Altogether, our data implicate an important unique function for Uhrf1 in neurogenesis and further research can indicate its relevance in the fields of reprogramming and repair.