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

Abstract

N-terminal methylation (Nα-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 Nα-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 Nα-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 Nα-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.

Opposing regulation of METTL11A by its family members METTL11B and METTL13

“…7D). We replicated previous findings that METTL11B significantly increased METTL11A methylation activity on the ZHX2 N-terminal peptide (4), but interestingly, found that inhibition by METTL13 takes precedence over activation by METTL11B. We found a significant decrease in ZHX2 methylation when both METTL11B and METTL13 are present compared to METTL11A alone (Fig.…”

“…Finally, as it appears all three family members can exist in a complex together, we were interested in determining if the regulatory effects of METTL13 or METTL11B took precedence when all three enzymes were present. As METTL11B has previously been shown to activate the activity of METTL11A specifically for non-canonical substrates (4,12), we used in vitro methyltransferase assays to measure if METTL13 also affected the ability of METTL11A to methylate the non-canonical substrate ZXH2. We found that, similar to the canonical RCC1 substrate (Fig.…”

“…Combined, the two consensus sequences predict over 300 METTL11A targets, and verified substrates include regulator of chromatin condensation 1 (RCC1), zinc fingers and homeoboxes 2 (ZHX2), and the ribosomal proteins RPS25 and RPL12 (1,4,5).…”

“…The non-canonical sequence expands to include one of seven amino acids other than Pro in the second position (A/S/G/M/E/N/Q), and either Lys or Arg in the third (3). Combined, the two consensus sequences predict over 300 METTL11A targets, and verified substrates include regulator of chromatin condensation 1 (RCC1), zinc fingers and homeoboxes 2 (ZHX2), and the ribosomal proteins RPS25 and RPL12 (1,4,5).…”

Opposing regulation of the Nα-trimethylase METTL11A by its family members METTL11B and METTL13

“…The noncanonical sequence expands to include one of seven amino acids other than Pro in the second position (A/S/G/M/E/N/Q) and either Lys or Arg in the third ( 3 ). Combined, the two consensus sequences predict over 300 METTL11A targets, and verified substrates include regulator of chromatin condensation 1 (RCC1), zinc fingers and homeoboxes 2 (ZHX2), and the ribosomal proteins RPS25 and RPL12 ( 1 , 4 , 5 ).…”