Biology subjects

Diehl, K. L.

Publications and source records attributed to Diehl, K. L..

4 recordsLinked to original sources

bioRxiv2024

Sirtuin 6 is a histone delactylase

Histone lactylation (Kla) is a post translational modification (PTM) that is derived from metabolic lactate. Histone Kla has been extensively studied in the field of inflammation resolution and macrophage polarization but has also been implicated in diverse cellular processes including differentiation, various wound repair phenotypes, and oncogenesis in several cancer models. While mechanistic connections between histone Kla and transcriptional changes have been studied in very limited contexts, general mechanistic details describing how regulation of gene expression by histone Kla occurs are scarce. It is hypothesized that histone Kla may be installed either through nonenzymatic means or by the p300 histone acyltransferase complex, and it is known that Class I HDACs and Sirtuins 1-3 can remove histone Kla. Here, we identified histone delactylase activity of the deacylase enzyme Sirtuin 6 (Sirt6), a member of the Class III HDAC family known to have roles in regulating metabolic homeostasis. We characterized the ability of Sirt6 to delactylate histones in vitro and in mammalian cell culture. We identified H3K9 and H3K18, canonical histone sites of Sirt6-catalyzed deacetylase activity, as sites of its delactylase activity. We also demonstrated that the delactylase activity of Sirt6 and the Class I HDACs are additive, suggesting that they represent different cellular axes of regulating gene expression via controlling levels of histone Kla.

biochemistry↗

bioRxiv2024

DNA stimulates SIRT6 to mono-ADP-ribosylate proteins within histidine repeats

Sirtuins are the NAD+-dependent class III lysine deacylases (KDACs). Members of this family have been linked to longevity and a wide array of different diseases, motivating the pursuit of sirtuin modulator compounds. Sirtuin 6 (SIRT6) is a primarily nuclear KDAC that deacetylates histones to facilitate gene repression. In addition to this canonical post-translational modification (PTM) "eraser" function, SIRT6 can use NAD+ instead to "write" mono-ADP-ribosylation (mARylation) on target proteins. This enzymatic function has been primarily associated with SIRT6s role in the DNA damage response. This modification has been challenging to study because it is not clear under what precise cellular contexts it occurs, only a few substrates are known, and potential interference from other ADP-ribosyltransferases in cells, among other reasons. In this work, we used commercially available ADP-ribosylation detection reagents to investigate the mARylation activity of SIRT6 in a reconstituted system. We observed that SIRT6 is activated in its mARylation activity by binding to dsDNA ends. We further identified a surprising target motif within biochemical substrates of SIRT6, polyhistidine (polyHis) repeat tracts, that are present in several previously identified SIRT6 mARylation substrates and binding partners. This work provides important context for SIRT6 mARylation activity, in contrast to its KDAC activity, and proposes that SIRT6 is a histidine mARyltransferase enzyme.

biochemistry↗

bioRxiv2024

A genetically-encoded fluorescent biosensor for visualization of acetyl-CoA in live cells

Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA production and consumption are highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. In this work, we engineer an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). We biochemically characterize the sensor and demonstrate its selectivity for acetyl-CoA over other CoA species. We then deploy the biosensor in E. coli and HeLa cells to demonstrate its utility in living cells. In E. coli, we show that the biosensor enables detection of rapid changes in acetyl-CoA levels. In human cells, we show that the biosensor enables subcellular detection and reveals the compartmentalization of acetyl-CoA metabolism.

biochemistry↗

bioRxiv2019

PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

Polycomb group (PcG) proteins are essential for development and are frequently misregulated in human cancers. Polycomb Repressive Complexes (PRC1, PRC2) function in a collaborative epigenetic cross-talk with H3K27me3 to initiate and maintain transcriptional silencing. Diffuse intrinsic pontine gliomas (DIPGs) have extremely low H3K27me3 levels mediated by H3 K27M oncohistone. Posterior fossa type A (PFA) ependymomas also exhibit very low H3K27 methylation but lack the K27M oncohistone. Instead, PFA tumors express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). We find that a highly conserved sequence within the C-terminus of EZHIP is necessary and sufficient to inhibit the catalytic activity of PRC2 in vitro and in vivo. Our biochemical experiments indicate that EZHIP directly interacts with the active site of the EZH2 subunit in a mechanism that is remarkably similar to the K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promote similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at the sites of PRC2 recruitment. Importantly, we find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a potential mechanism for loss of H3K27me3 spreading from CpG islands in vivo. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors- the K27M oncohistone and the EZHIP oncohistone-mimic- that dysregulate gene silencing to promote tumorigenesis.

cancer biology↗