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The role of chromatin associated protein HMGB2 in setting up permissive chromatin states for direct glia to neuron conversion
The role of chromatin associated protein HMGB2 in setting up permissive chromatin states for direct glia to neuron conversion
The adult mammalian brain has a limited capacity to replace the loss of neurons following the traumatic brain injury or disease, therefore requiring innovative strategies to promote tissue regeneration and functional repair of the central nervous system (CNS). Direct lineage reprogramming of non-neuronal cell types (reactive astrocytes and oligodendrocytes), resident within the injured CNS to generate new neurons is a promising approach to repair damaged brain. Despite very good conversion rate of isolated postnatal astrocytes in vitro and recent advances to produce induced neuronal cells in vivo, the molecular understanding of the reprogramming process remains largely unknown. Toward this end, we developed an in vitro model with adjusted growth factor composition lacking epidermal growth factor (EGF) that reduces the reprogramming efficiency of astroglia into neurons to the rate similar to reactive astrocytes in vivo. By comparing the cultures prone and resistant to reprogramming we aimed to identify molecular features required for the efficient conversion. The proteome analysis of EGF+FGF and FGF cultures revealed the chromatin state changes as the main factor that could maintain the astrocytes in the glial lineage even after neurogenic factor overexpression. To test this hypothesis, we overexpressed the most regulated chromatin-related protein, HMGB2, together with NEUROG2 and analysed the reprogramming efficiency. Indeed, the simultaneous overexpression of both factors in the astrocytes resistant to the fate transition significantly increased direct reprogramming suggesting the role of chromatin-associated proteins in bypassing the lineage roadblocks. To understand the role of global chromatin rearrangement, we performed ATAC-sequencing comparing both culture conditions and searched for HMGB2 induced changes in the chromatin compaction. The chromatin accessibility analysis revealed an enrichment of opened promoters assigned to the neuronal genes in the astrocytes with good reprogramming capacity and the ability of HMGB2 to open these specific promoters in the culture resistant to reprogramming. In order to access the transcriptional changes induced by HMGB2 and NEUROG2 overexpression, we performed RNA-sequencing that indicated the involvement of HMGB2 in repression of the myogenic alternative fate, possibly by the opening the repressor binding sites. Taken together, we identified the novel candidate HMGB2 in the prospect of direct neuronal reprogramming making the chromatin states in the glial cells permissive for lineage conversion and thereby enabling acquisition of the neuronal phenotype in the cultures with limited reprogramming capacity.
Not available
Lepko, Tjaša
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Lepko, Tjaša (2018): The role of chromatin associated protein HMGB2 in setting up permissive chromatin states for direct glia to neuron conversion. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)
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Abstract

The adult mammalian brain has a limited capacity to replace the loss of neurons following the traumatic brain injury or disease, therefore requiring innovative strategies to promote tissue regeneration and functional repair of the central nervous system (CNS). Direct lineage reprogramming of non-neuronal cell types (reactive astrocytes and oligodendrocytes), resident within the injured CNS to generate new neurons is a promising approach to repair damaged brain. Despite very good conversion rate of isolated postnatal astrocytes in vitro and recent advances to produce induced neuronal cells in vivo, the molecular understanding of the reprogramming process remains largely unknown. Toward this end, we developed an in vitro model with adjusted growth factor composition lacking epidermal growth factor (EGF) that reduces the reprogramming efficiency of astroglia into neurons to the rate similar to reactive astrocytes in vivo. By comparing the cultures prone and resistant to reprogramming we aimed to identify molecular features required for the efficient conversion. The proteome analysis of EGF+FGF and FGF cultures revealed the chromatin state changes as the main factor that could maintain the astrocytes in the glial lineage even after neurogenic factor overexpression. To test this hypothesis, we overexpressed the most regulated chromatin-related protein, HMGB2, together with NEUROG2 and analysed the reprogramming efficiency. Indeed, the simultaneous overexpression of both factors in the astrocytes resistant to the fate transition significantly increased direct reprogramming suggesting the role of chromatin-associated proteins in bypassing the lineage roadblocks. To understand the role of global chromatin rearrangement, we performed ATAC-sequencing comparing both culture conditions and searched for HMGB2 induced changes in the chromatin compaction. The chromatin accessibility analysis revealed an enrichment of opened promoters assigned to the neuronal genes in the astrocytes with good reprogramming capacity and the ability of HMGB2 to open these specific promoters in the culture resistant to reprogramming. In order to access the transcriptional changes induced by HMGB2 and NEUROG2 overexpression, we performed RNA-sequencing that indicated the involvement of HMGB2 in repression of the myogenic alternative fate, possibly by the opening the repressor binding sites. Taken together, we identified the novel candidate HMGB2 in the prospect of direct neuronal reprogramming making the chromatin states in the glial cells permissive for lineage conversion and thereby enabling acquisition of the neuronal phenotype in the cultures with limited reprogramming capacity.