Schaner Tooley, C. | bioRxiv Science

bioRxiv2021

Novel regulation of the transcription factor ZHX2 by N-terminal methylation

N-terminal methylation (N-methylation) by the methyltransferase NRMT1 is an important post-translational modification that regulates protein-DNA interactions. Accordingly, its loss impairs functions that are reliant on such interactions, including DNA repair and transcriptional regulation. Global loss of N-methylation results in severe developmental and premature aging phenotypes, but given over 300 predicted substrates, it is hard to discern which physiological substrates contribute to each phenotype. One of the most striking phenotypes in NRMT1 knockout (Nrmt1-/-) mice is early liver degeneration. To identify the disrupted signaling pathways leading to this phenotype and the NRMT1 substrates involved, we performed RNA-sequencing analysis of control and Nrmt1-/- adult mouse livers. We found both a significant upregulation of transcripts in the cytochrome P450 (CYP) family and downregulation of transcripts in the major urinary protein (MUP) family. Interestingly, transcription of both families is inversely regulated by the transcription factor zinc fingers and homeoboxes 2 (ZHX2). ZHX2 contains a non-canonical NRMT1 consensus sequence, indicating its function could be directly regulated by N-methylation. We confirmed misregulation of CYP and MUP mRNA and protein levels in Nrmt1-/- livers and verified NRMT1 can methylate ZHX2 in vitro. In addition, we used mutants of ZHX2 that cannot be methylated to directly demonstrate N-methylation promotes ZHX2 transcription factor activity. Finally, we show Nrmt1-/- mice also exhibit early postnatal de-repression of ZHX2 targets involved in fetal liver development. Taken together, these data implicate continual ZHX2 misregulation as a driving force behind the liver phenotype seen in Nrmt1-/- mice.

biochemistry↗

bioRxiv2021

Age-related neurodegeneration and cognitive impairments of NRMT1 knockout mice are preceded by misregulation of RB and expansion of the neural stem cell population

N-terminal methylation is an important post-translational modification that regulates protein/DNA interactions and plays a role in many cellular processes, including DNA damage repair, mitosis, and transcriptional regulation. Our generation of a constitutive knockout mouse for the N-terminal methyltransferase NRMT1, demonstrated its loss results in severe developmental abnormalities and premature aging. As premature aging is often accompanied by neurodegeneration, we more specifically examined how NRMT1 loss affects neural pathology and cognitive behaviors. Here we find that Nrmt1-/- mice exhibit postnatal enlargement of the lateral ventricles, age-dependent striatal and hippocampal neurodegeneration, memory impairments, and hyperactivity. These morphological and behavior abnormalities are preceded by alterations in neural stem cell (NSC) development. Depletion of quiescent NSC pools in Nrmt1-/- mice is concurrent with expansion of intermediate progenitor and neuroblast pools. These phenotypes are similar to those seen with loss of the NRMT1 target retinoblastoma protein (RB), and we see that NRMT1 loss leads to derepression of RB target genes and abnormal RB phosphorylation and degradation. As also seen with RB loss, neurons in Nrmt1-/- mice fail to exit cell cycle and ultimately undergo NOXA-mediated apoptosis, indicating that early misregulation of RB in Nrmt1-/- mice promotes premature NSC proliferation and contributes to subsequent neurodegenerative phenotypes.

neuroscience↗