Biology subjects

Hinchcliffe, E. H.

Publications and source records attributed to Hinchcliffe, E. H..

3 recordsLinked to original sources

bioRxiv2024

Commitment to cytokinetic furrowing requires the coordinate activity of microtubules and Plk1

At anaphase, spindle microtubules (MTs) position the cleavage furrow and trigger actomyosin assembly by localizing the small GTPase RhoA and the scaffolding protein anillin to a narrow band along the equatorial cortex [1-6]. Using vertebrate somatic cells we examined the temporal control of furrow assembly. Although its positioning commences at anaphase onset, furrow maturation is not complete until [~]10-11 min later. The maintenance of the RhoA/anillin scaffold initially requires continuous signaling from the spindle; loss of either MTs or polo-like kinase 1 (Plk1) activity prevents proper RhoA/anillin localization to the equator, thereby disrupting furrowing. However, we find that at [~]6 min post-anaphase, the cortex becomes "committed to furrowing"; loss of either MTs or Plk1 after this stage does not prevent eventual furrowing, even though at this point the contractile apparatus has not fully matured. Also at this stage, the RhoA/anillin scaffold at the equator becomes permanent. Surprisingly, concurrent loss of both MTs and Plk1 activity following the "commitment to furrowing" stage results in persistent, asymmetric "half-furrows", with only one cortical hemisphere retaining RhoA/anillin, and undergoing ingression. This phenotype is reminiscent of asymmetric furrows caused by a physical block between spindle and cortex [7-9], or by acentric spindle positioning [10-12]. The formation of these persistent "half-furrows" suggests a potential feedback mechanism between the spindle and the cortex that maintains cortical competency along the presumptive equatorial region prior to the "commitment to furrowing" stage of cytokinesis, thereby ensuring the eventual ingression of a symmetric cleavage furrow.

cell biology↗

bioRxiv2024

H3F3A K27M Mutations Drives a Repressive Transcriptome by Modulating Chromatin Accessibility, Independent of H3K27me3 in Diffuse Midline Glioma

BackgroundHeterozygous histone H3.3K27M mutation is a primary oncogenic driver of Diffuse Midline Glioma (DMG). H3.3K27M inhibits the Polycomb Repressive Complex 2 (PRC2) methyltransferase complex, leading to a global reduction and redistributing of the repressive H3 lysine 27 tri-methylation. This rewiring of the epigenome is thought to promote gliomagenesis. MethodsWe established novel, isogenic DMG patient-derived cell lines that have been CRISPR-Cas9 edited to H3.3 WT or H3.3K27M alone and in combination with EZH2 and EZH1 co-deletion, inactivating PRC2 methyltransferase activity of PRC2 and eliminating H3K27me3. ResultsRNA-seq and ATAC-seq analysis of these cells revealed that K27M has a novel epigenetic effect that appears entirely independent of its effects on PRC2 function. While the loss of the PRC2 complex led to a systemic induction of gene expression (including HOX gene clusters) and upregulation of biological pathways, K27M led to a balanced gene deregulation but having an overall repressive effect on the biological pathways. Importantly, the genes uniquely deregulated by the K27M mutation, independent of methylation loss, are closely associated with changes in chromatin accessibility, with upregulated genes becoming more accessible. Notably, the PRC2- independent function of K27M appears necessary for tumorigenesis as xenografts of our H3.3K27M/EZH1/2 WT cells developed into tumors, while H3.3/EZH1/2 KO cells did not. ConclusionWe demonstrate that K27M mutation alters chromatin accessibility and uniquely deregulates genes, independent of K27 methylation. We further show the mutations role in altering biological pathways and its necessity for tumor development. Key PointsO_LIWe revealed genes regulated by H3.3K27M mutation and PRC2 in DMG. C_LIO_LIH3.3K27M mutation alters chromosome accessibility independent of H3K27me3. C_LIO_LIPRC2-independent effects of K27M mutation are crucial for tumor development. C_LI Importance of the StudyThis study is the first to demonstrate that H3F3A K27M mutations drive a repressive transcriptome by modulating chromatin accessibility independently of H3K27 trimethylation in Diffuse Midline Glioma (DMG). By isolating the effects of H3.3 K27me3 loss from those of the K27M mutation, we identified common and unique genes and pathways affected by each. We found that genes uniquely deregulated by K27M showed increased chromatin accessibility and upregulated gene expression, unlike other gene subsets affected by PRC2 knockout. Importantly, we determined the PRC2-independent function of K27M is also essential for tumorigenesis, as xenografts of H3.3 K27M/PRC2 WT cell lines formed tumors, while H3.3WT/PRC2 WT and K27M/PRC2 knockout cells did not. This research builds upon and advances prior studies, such as those identifying EZH2 as a therapeutic target in H3.3K27M DMGs, by revealing critical new pathways for gliomagenesis. The translational significance lies in identifying novel therapeutic targets against this aggressive pediatric cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/594522v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@61edd6org.highwire.dtl.DTLVardef@145935dorg.highwire.dtl.DTLVardef@cde85aorg.highwire.dtl.DTLVardef@5184b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

The histone H3.3 K27M mutation found in diffuse midline gliomas coordinately disrupts adjacent H3.3 Ser31 phosphorylation and the fidelity of chromosome segregation

During the cell cycle, differential phosphorylation of select histone H3 serine/threonine residues regulates chromatin structure, necessary for both dynamic transcriptional control and proper chromosome segregation1-2. Histone H3.3 contains a highly conserved serine residue (Ser31) within its N-terminal tail that is unique to this variant. During interphase phosphorylation of Ser31 amplifies stimulation-induced transcription and is required for early metazoan development3-6. During mitosis Ser31 phosphorylation at the pericentromere supports proper chromosome segregation, albeit by unknown mechanisms7-10. H3.3 Ser31 is flanked by mutational sites that drive several human cancers, including pediatric gliomas5-8. This is typified by the H3.3K27M mutation found in [~]80% of diffuse midline gliomas, which undergo epigenetic reprogramming in proliferative cells coordinate with loss of global H3 lysine 27 trimethylation (H3K27Me3)11-14. However, whether the K27M mutation influences the neighboring Ser31 phosphorylation and whether disrupting Ser31 phosphorylation plays a distinct role in driving gliomagenesis has not been tested. Here we show that H3.3K27M mutant cells have reduced capacity for H3.3 Ser31 phosphorylation at the mitotic pericentromere, increased rates of chromosome missegregation, and impaired G1 checkpoint responses to chromosome instability. CRISPR-reversion of K27M to wild-type restores phospho-Ser31 levels and suppresses chromosome segregation defects. CRISPR editing to introduce a non-phosphorylatable H3.3S31A alone is sufficient to increase the frequency of chromosome missegregations. Finally, expression of H3.3S31A in a PDGF{beta}-driven RCAS/TVA mouse model is sufficient to drive high grade gliomagenesis, generating diffuse tumors morphologically indistinguishable from those generated by H3.3K27M expression. Importantly, this occurs without the loss of H3K27 triple methylation that is the hallmark of K27M tumors. Our results reveal that the H3.3 K27M mutation alters the neighboring Ser31 phosphorylation, and loss of proper H3.3 Ser31 phosphorylation contributes to the formation of diffuse midline gliomas.

cancer biology↗