The proposed research will examine how chromatin regulates gene expression during CNS migration and circuit formation, using the cerebellar granule cell as a model. The work builds on our recent discovery that dramatic changes occur in the levels of chromatin remodeling genes during cerebellar development, including changes in histone modifying genes and in Tet genes, which oxidize 5-methyl- cytosine (5mC) into 5-hydroxymethyl-cytosine (5hmC), a brain-specific DNA modification. Our studies showed that increased levels of 5hmC correlated with increased levels of ion channel genes and of axon guidance (dendritic) genes in post-migratory neurons (Zhu et al, 2016). Moreover, RNAi-mediated knockdown of Tet genes in migrating cerebellar granule cells in ex vivo tissue slices blocked the transition from a bipolar, migrating cell into a multipolar cell that is extending dendrites and forming synaptic contacts with ingrowing afferent, mossy fibers (Zhu et al, 2016). These findings provided the first evidence for the idea that chromatin changes underlie the formation of the cerebellar circuitry. In the proposed research, we will collaborate with our colleagues, Dr. David Allis and Dr. Erica Korb, who are authorities on chromatin biology, to characterize modifications in four basic histone methylation marks that regulate transcriptional activation and repression, histone 3 lysine residue 4 (H3K4), H3K9, H3K27 and H3K36, in a well-characterized CNS neuron, the cerebellar granule cell (GC), before, during and just after glial-guided migration, as the cerebellar circuitry forms. We will then identify changes in gene expression associated with these histone methylation changes. Finally, we will use RNAi methodology to study the function of histone methyltransferases and demethylases involved in generating these histone marks in migration, dendrite extension and synapse formation on ex vivo cerebellar slices. These studies will provide critical information on chromatin changes underlying CNS migration and circuit formation in the cerebellum.
The proposed research will provide key information on how chromatin regulates gene expression during brain development. It will provide critical new insights into the molecular basis of the formation of the cerebellar circuitry, including insights into developmental brain disorders.
