Biology subjects

Strich, R.

Publications and source records attributed to Strich, R..

5 recordsLinked to original sources

bioRxiv2026

Cyclin C nuclear release and mitochondrial dysfunction define molecular signatures of MED13L Syndrome

The Mediator Kinase Module (MKM) coordinates transcriptional programs regulating cellular metabolism, stress responses, and differentiation. Heterozygous variants of MED13L, a core MKM component, cause a neurodevelopmental disorder characterized by variable intellectual disability, developmental delay, neuromuscular dysfunction, and congenital anomalies. However, the molecular basis underlying this clinical heterogeneity is poorly defined. Previously, we identified mitochondrial dysfunction and aberrant nuclear release of another MKM component, cyclin C (CCNC), in a single MED13L syndrome patient-derived fibroblast line. Here, we expand these studies across 12 patient-derived fibroblasts harboring 11 distinct MED13L variants. We identify mitochondrial dysfunction as a consistent feature of MED13L variation, characterized by reduced mitochondrial ATP production, decreased mitochondrial DNA abundance, elevated reactive oxygen species, and impaired transcription of genes involved in mitochondrial biogenesis. In parallel, all variant lines exhibit aberrant cytoplasmic CCNC localization, consistent with its established role in mitochondrial fission. Longitudinal analyses further reveal progressive declines in mitochondrial function associated with premature cellular aging, consistent with increased metabolic deficits. Importantly, the severity of mitochondrial dysfunction shows an association with variant position within MED13L and with clinical functional measures, suggesting that mutation location may partially predict disease severity. Together, these findings establish mitochondrial dysfunction as a consistent cellular feature of MED13L heterozygosity and identify CCNC mis-localization as a candidate biomarker of MKM disruption. More broadly, this work reveals an intersection between transcriptional control and mitochondrial homeostasis in MED13L syndrome, forming the framework for biomarker-driven therapeutic development in MED13L-associated and related neurodevelopmental disorders.

genetics↗

bioRxiv2025

A conserved mitochondrial role for cyclin C in mediating oxidative stress-induced cell death in yeast

Cyclin C is a highly conserved component of the Mediator kinase module that regulates transcription through CDK8 stimulation. In addition, the yeast (Cnc1) and human (CCNC) cyclin C exhibit stress-induced mitochondrial translocation to stimulate fission through direct interaction with the dynamin-like GTPase DRP1 (mammals) or Dnm1 (yeast). Gene ablation studies revealed that both Cnc1 and CCNC are required for cell damage-induced regulated cell death (RCD). To determine the relative contributions of cyclin Cs transcriptional and mitochondrial roles in promoting RCD, this study utilized docking simulation algorithms to predict interaction interfaces for CCNC-CDK8 and CCNC-DRP1 heterodimers. As expected, CCNC bound CDK8 through its amino terminal cyclin box domain while DRP1 associated with the second carboxyl cyclin box. Using these predictions, we used site directed mutagenesis on Cnc1 to separate these functions. Importantly, only the DRP1/Dnm1-interaction residues were important for RCD in yeast. Interestingly, although Dnm1 is required for RCD, its fission activity was not. Moreover, Dnm1 is still required for RCD even when Cnc1 is targeted to the mitochondria indicating it is not simply functioning as a tether. Finally, when expressed in yeast, the human CCNC efficiently induced fission and stimulated RCD. Moreover, these functions required predicted DRP1 interaction sites as well. In conclusion, these studies revealed that cyclin C separates its nuclear and mitochondrial activities by utilizing different cyclin box domains. Second, Dnm1-cyclin C interaction, and not transcriptional control, is critical for cyclin C-dependent RCD and this role is conserved from yeast to humans. Author SummaryThe cyclin C protein is found in all eucaryotes and exhibits two conserved functions. First cyclin C activates the CDK8 protein kinase to control transcription of genes involved in the stress response. Second, stress induces cyclin C translocation to the mitochondria where it induces fragmentation by stimulating the fission GTPase DRP1. Mitochondrial fission is an early step in the regulated cell death pathway. Importantly, cyclin C is required for stress-induced cell death raising the question of what the relative contribution of its transcription and mitochondrial roles is. To address this question, we generated mutations in the yeast cyclin C that interfered with its association with CDK8 or DRP1 without affecting the other. We found that the mitochondrial role, but not transcription, was required for cell death in yeast. In addition, when expressed in yeast, the human cyclin C also induced mitochondrial fission and cell death. Finally, although DRP1 is important for regulated cell death, its fission function is not. These results point to a highly conserved role for cyclin C in mediating regulated cell death at the mitochondria. In addition, these results point to a non-enzymatic role for DRP1 in executing the cell death pathway.

cell biology↗

bioRxiv2024

Cyclin C Promotes Pancreatic Development and Suppresses Cancer Initiation Through Maintenance of the Autophagy-Lysosome Pathway

The cyclin C (Ccnc)-Cdk8/Cdk19 kinases are components of the Mediator that represses or activates gene transcription. The present study found that Ccnc is required for both steady state and induced autophagic gene transcription in mouse embryonic fibroblasts. In vivo, pancreatic ablation of Ccnc (CcncPan{Delta}) resulted in phenotypes (islet atrophy, acinar cell damage) also observed in autophagy deficient models. However, CcncPan{Delta}animals displayed more dramatic phenotypes including earlier death and accelerated acinar ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) precancerous lesion formation when these animals also expressed oncogenic KrasG12D. Consistent with the in vivo results, a KrasG12D; CcncPan{Delta} pancreatic derived cell line displayed reduced autophagy lysosome pathway (ALP) activation. Although autophagy deficient acinar cells undergo Tp53-dependent cell death, histopathology revealed that KrasG12D;Ccnc-/- (PC) pancreata did not allowing damaged cells to keep dividing. Therefore, Ccnc both supports normal ALP function protecting endocrine and exocrine cell but also kills damaged cells before they become malignant. Finally, the PC cell line displayed reduced proteasome function rendering cells hypersensitive to proteasome inhibitors. This hypersensitivity was also observed in disparate Ccnc-/- tumor cell lines or in Ccnc+/+ tumor cells co-treated with Cdk8/Cdk19 inhibitors. These findings suggest a new avenue to target pancreatic neoplasms by inhibiting cyclin C-Cdk8/Cdk19 proteasome activity.

developmental biology↗

bioRxiv2022

An in situ cut-and-paste genome editing platform mediated by CRISPR/Cas9 or Cas12a

Recombinant DNA technology mediated by restriction enzymes and ligases allows in vitro manipulation of a DNA segment isolated from the genome. Short overhangs generated by restriction enzymes facilitate efficient pasting together a DNA sequence and a vector. We adopted this recombinant DNA strategy to develop an in vivo recombinant-genome genome editing approach. Using the programmable endonuclease Cas9 or Cas12a as a restriction enzyme, we devised an in situ cut-and-paste (iCAP) genome editing method that was tested in both mouse germline and human cell line platforms. Mouse gene loci Slc35f2 and Slc35f6 were each edited with in-frame insertion of a large APEX2-Cre cassette and concurrent FRT3 insertion at a second location providing proof of principle for the iCAP method. Further, a de nova single nucleotide mutation associated with MED13L syndrome was efficiently corrected in patient cells. Altogether, the iCAP method provides a single genome editing platform with flexibility and multiutility enabling versatile and precise sequence alterations, such as insertion, substitution, and deletion, at single or multiple locations within a genomic segment in mammalian genomes.

molecular biology↗

bioRxiv2020

Cyclin C promoter occupancy directs changes in stress-dependent transcription

The Cdk8 kinase module (CKM) is a detachable Mediator subunit composed of cyclin C, and one each of paralogs Cdk8/Cdk19, Med12/Med12L and Med13/Med13L. In addition to regulating transcription, a portion of cyclin C also leaves the nucleus following cytotoxic stress to induce mitochondrial fragmentation and apoptosis. Our previous RNA-seq studies demonstrated that cyclin C represses a subset of hydrogen peroxide-induced genes under normal conditions, while also being required for the full induction of other loci following stress. Here, we show that cyclin C directs this transcriptional reprograming through changes in its promoter occupancy. Following peroxide stress, cyclin C promoter occupancy increased for genes it activates while decreasing at loci it represses under normal conditions. Promoter occupancy of other CKM components generally mirrored cyclin C indicating that the CKM moves as a single unit. However, CKM integrity appeared compromised at a subset of repressed promoters suggesting a source of cyclin C that is targeted for nuclear release. Interestingly, mTOR inhibition induced a new pattern of cyclin C promoter occupancy indicating that this control is fine-tuned to the individual stress. Using inhibitors, we found that Cdk8 kinase activity is not required for CKM movement or repression but was necessary for full gene activation. In conclusion, this study revealed that different stress stimuli elicit specific changes in CKM promoter occupancy correlating to altered transcriptional outputs. Finally, although CKM components were recruited or expelled from promoters as a unit, heterogeneity was observed at individual promoters suggesting a mechanism to generate gene- and stress-specific responses.

molecular biology↗