Biology subjects

Panning, B.

Publications and source records attributed to Panning, B..

9 recordsLinked to original sources

bioRxiv2026

Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

Repressive histone methyltransferases carry a catalytic ("write") domain and a separate domain specialized for recognizing ("reading") the reaction product. This read-write configuration acts as a positive feedback mechanism for epigenetic maintenance and the growth of repressive chromatin domains. Feedback exhibits as catalytic stimulation and is understood to act towards a proximal (trans) nucleosome. Whether this stimulation affects a specific methylation transition and whether it is restricted to trans-stimulation remains opaque. Here, we dissect the positive feedback in the heterodimeric histone 3 lysine 9 (H3K9) mono- and dimethlyase G9a-GLP, which carries two catalytic SET and two product-reading Ankyrin repeat (ANK) domains. We find that reading by both ANK domains is required for H3K9 di-, but not monomethylation on nucleosomes and for tight binding to them. As this read-writing occurs on dilute mononucleosomes, we propose that intranucleosomal feedback occurs for G9a-GLP. Swapping the ANK domains results in loss of dimethylation while maintaining nucleosome binding, indicating catalytic coupling of nucleosome methylation intermediates to reading. Crosslinking mass spectrometry reveals specific G9a surfaces that contact nucleosomal methylation intermediates. Structural approaches reveal how these surfaces position the G9a ANK domain on the methylation-intermediate nucleosome and stabilize G9a-GLP on chromatin during the reaction.

biochemistry↗

bioRxiv2025

O-GlcNAc modifications regulate lamin A tail processing

Lamin A processing is highly regulated, and necessary for proper assembly of the nuclear lamina facilitating its role in nuclear structure and chromatin organization. Pre-lamin A is first farnesylated, and then a short C-terminal peptide is cleaved to produce mature lamin A. O-GlcNAc Transferase (OGT), a glucose sensitive post-translational modification enzyme, is a potential regulator for lamin A processing. To explore the role of OGT in lamin A biogenesis, we examined the effects of OGT levels and OGT inhibition. Variation in OGT dose or inhibition of its activity did not alter endogenous lamin A abundance or distribution. To more directly test the regulatory effects of O-GlcNAcylation on lamin A, we adapted a tail cleavage assay. Mutation of an OGT binding motif and O-GlcNAc modification sites reduced tail cleavage efficiency, suggesting that O-GlcNAcylation promotes lamin A processing. Our findings add to the understanding of the regulation of lamin A cleavage and identify a potential link between glucose metabolism and lamina biogenesis.

cell biology↗

bioRxiv2024

The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate

Heterochromatic loci marked by histone H3 lysine 9 dimethylation (H3K9me2) are enriched at the nuclear periphery in metazoans, but the effect of spatial position on heterochromatin function has not been defined. Here, we remove three nuclear lamins and lamin B receptor (LBR) in mouse embryonic stem cells (mESCs) and show that heterochromatin detaches from the nuclear periphery. Mutant mESCs sustain naive pluripotency and maintain H3K9me2 across the genome but cannot repress H3K9me2-marked genes or transposons. Further, mutant cells fail to differentiate into epiblast-like cells (EpiLCs), a transition that requires the expansion of H3K9me2 across the genome. Mutant EpiLCs can silence naive pluripotency genes and activate epiblast-stage genes. However, H3K9me2 cannot repress markers of alternative fates, including primitive endoderm. We conclude that the nuclear periphery controls the spatial position, dynamic remodeling, and repressive capacity of H3K9me2-marked heterochromatin to shape cell fate decisions.

cell biology↗

bioRxiv2022

X chromosome factor Kdm6a enhances memory independent of its demethylase function in the aging XY male brain

Males exhibit shorter lifespan and more cognitive deficits in aging human populations. In mammals, the X chromosome is enriched for neural genes and is a major source of biologic sex difference, in part, because males show decreased expression of select X factors. While each sex (XX and XY) harbors one active X due to X chromosome inactivation in females, some genes, such as Kdm6a, transcriptionally escape silencing in females - resulting in lower levels in males. Kdm6a is a known histone demethylase (H3K27me2/3) with multiple functional domains that is linked with synaptic plasticity and cognition. Whether elevating Kdm6a could benefit the aging male brain and whether this requires its demethylase function remains unknown. We used lentiviral-mediated overexpression of the X factor in the hippocampus of aging male mice and tested their cognition and behavior in the Morris water maze. We found that acutely increasing Kdm6a - in a form without demethylase function - selectively improved learning and memory, without altering total activity or anxiety-like measures, in the aging XY brain. Further understanding the demethylase-independent downstream mechanisms of Kdm6a may lead to novel therapies for treating age-induced cognitive deficits in both sexes.

neuroscience↗

bioRxiv2022

The maternal X chromosome impairs cognition and accelerates brain aging through epigenetic modulation in female mice

Female mammalian cells harbor two X chromosomes, one of maternal and one of paternal origin. During development, one X randomly inactivates1-4. This renders either the maternal or paternal X active, causing X mosaicism that varies among individual females, with some showing considerable or complete skew in the general female population5-7. Parent-of-X-origin can modify epigenetics via DNA methylation8, 9 and possibly gene expression; thus, mosaicism could buffer dysregulated processes in aging and disease. However, whether X skewing - or its mosaicism - alters functions in females is largely unknown. Here we tested whether skew toward the maternal X (Xm) influences key functions of the body. Among cardiac, bone, metabolic, and brain functions, Xm selectively impaired cognition in female mice throughout the lifespan. Cognitive deficits were accompanied by Xm-mediated acceleration of biologic or epigenetic aging of the female hippocampus, a key center for learning and memory. Xm showed epigenetic imprinting of several genes within hippocampal neurons, suggesting silenced cognitive loci. Thus, the maternal X chromosome impaired cognition, accelerated brain aging, and silenced genes. Understanding how the maternal X impairs brain function could lead to new understanding of female heterogeneity in cognitive heath and to X chromosome-derived pathways against cognitive deficits and brain aging.

neuroscience↗

bioRxiv2021

A dose-sensitive OGT-TET3 complex is necessary for normal Xist RNA distribution and function

Female (XX) mouse embryonic stem cells (mESCs) differ from their male (XY) counterparts because they have lower levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). This difference in DNA modifications is a result of having two X chromosomes (Xs), both of which are active at this developmental stage. We identified an X-linked gene, Ogt, that controls levels of 5mC and 5hmC in mESCs. OGT is a post-translational modification enzyme and we identified the 5-methylcytosine dioxygenase TET3 as an OGT target that is differentially modified in XX and XY mESCs. In addition to influencing 5mC and 5hmC abundance, OGT dose also controls TET3 and OGT distribution. OGT and TET3 are predominantly nuclear in XX mESCs and cytoplasmic in XY mESCs. Furthermore, these proteins are present in different complexes in XX and XY mESCs. Mutational analysis revealed that TET3 determines the XX-specific abundance of 5mC and 5hmC in mESCs. While TET3 null XX mESCs exhibited modest changes in gene expression, there were substantial alterations upon differentiation into epiblast-like cells (mEpiLCs). In addition, these TET3 null XX mESCs did not undergo X-chromosome inactivation (XCI) when differentiated. These data suggest that an X-dose sensitive complex containing OGT and TET3 regulates cytosine modifications and XCI.

genetics↗

bioRxiv2019

Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.

genomics↗

bioRxiv2018

OGT binds a conserved C-terminal domain of TET1 toregulate TET1 activity and function in development

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a domain of TET1 responsible for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that the TET1-OGT interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1s function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET-containing high molecular weight complexes and causes widespread gene expression changes. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

biochemistry↗

bioRxiv2017

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development

Mammalian TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs are targets of the enzyme OGT, which post-translationally modifies intracellular proteins in response to cellular nutrient status. The biological implications of the OGT-TET interaction have not been thoroughly explored. Here, we show for the first time that modification of TET1 by OGT enhances its activity in vitro. We identify a previously uncharacterized domain of TET1 responsible for binding to OGT and report a point mutation that disrupts the OGT-TET1 interaction. Finally, we show that the interaction between TET1 and OGT is necessary for TET1 to rescue tet mutant zebrafish hematopoetic stem cell formation, suggesting that OGT promotes TET1s function in development. Our results demonstrate regulation of TET activity by OGT in vitro and in vivo. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

biochemistry↗