Biology subjects

Khokha, M. K.

Publications and source records attributed to Khokha, M. K..

7 recordsLinked to original sources

bioRxiv2025

Equine Parentage Testing with SNPs and beyond: developing a platform independent panel

Centuries of selective equine breeding have led to a remarkable diversity of horse breeds each with unique traits. To maintain these breeds, studbooks require parentage testing to guarantee lineage. To determine parentage, the industry assays length polymorphisms in short tandem repeats (STRs) established by the International Society of Animal Genetics (ISAG). However, STRs have significant limitations including challenges with accurately determining genotype, relatively low number of STRs assayed, and low throughput. For these reasons, most other animal breeds including cattle, dog, goats, and sheep use single nucleotide polymorphisms (SNPs) for parentage identification. To this end, the ISAG has introduced a panel of SNPs. Here, we further demonstrate the utility of equine SNP genotyping. Importantly, we show that a number of the ISAG SNPs are not compatible with widely-used, short-read, next generation sequencing (NGS) technologies. Fortunately, SNPs are abundant, and we propose an alternative panel that is platform independent and are all ISAG proposed SNPs. We demonstrate that SNP parentage genotyping is extremely robust and enables additional assays including breed identification and can be readily expanded to also assay equine genetic diseases.

genomics↗

bioRxiv2025

A yeast model reveals the SMAD3 nuclear import mechanism

TGF-{beta} signaling is important for patterning tissues during embryonic development and relies on the nuclear import of SMADs. Basal SMAD nuclear import mechanisms remain to be fully understood and could benefit from advances in our understanding of nuclear transport machinery. To assess the molecular determinants of SMAD nuclear transport, we take advantage of the unicellular S. cerevisiae model that lacks the TGF-{beta} signaling complexity of vertebrates but has conserved nuclear transport machinery. In this minimal system, we find that the steady-state distribution of SMAD2/3, SMAD4, and SMAD1/5 are Ran-dependent and either accumulate in, or are depleted from the nucleus. Conditionally inhibiting each of the karyopherin/importins demonstrates that SMAD3 is imported by orthologues of importin-{beta}, importin-7 and importin-8. The previously defined nuclear localization signal is insufficient to confer nuclear import. Instead, our data suggest the entire MH1 domain may act as an importin binding surface. Deleting a portion of this domain impairs SMAD3 function in Xenopus and SMAD3 nuclear enrichment in HEK293T cells. Thus, the yeast platform provides an efficient strategy to illuminate the nuclear transport mechanisms of embryonic signaling effectors.

developmental biology↗

bioRxiv2022

Expanding EMC Foldopathies: Topogenesis Deficits Alter the Neural Crest

The endoplasmic reticulum membrane protein complex (EMC) is essential for the insertion of a wide variety of transmembrane proteins into the plasma membrane across cell types. Each EMC is composed of Emc1-7, Emc10, and either Emc8 or Emc9. Recent human genetics studies have implicated variants in EMC genes as the basis for a group of human congenital diseases. The patient phenotypes are varied but appear to affect a subset of tissues more prominently than others. Namely, craniofacial development seems to be commonly affected. We previously developed an array of assays in Xenopus tropicalis to assess the effects of emc1 depletion on the neural crest, craniofacial cartilage, and neuromuscular function. We sought to extend this approach to additional EMC components identified in patients with congenital malformations. Through this approach we determine that EMC9 and EMC10 are important for neural crest development and the development of craniofacial structures. The phenotypes observed in patients and our Xenopus model were similar to EMC1 loss of function likely due to a similar mechanism of dysfunction in transmembrane protein topogenesis.

developmental biology↗

bioRxiv2021

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs

Frogs are an ecologically diverse and phylogenetically ancient group of living amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., centric) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding, surrounded by pericentromeric LINE/L1 elements. We explored chromosome structure across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (HiC) data for all species. Abundant satellite repeats occupy the unusually long ([~]20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible association of centromeric chromatin, and of telomeres, reflecting a Rabl configuration similar to the "bouquet" structure of meiotic cells. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

genomics↗

bioRxiv2017

CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.

genetics↗

bioRxiv2017

WDR5 stabilizes actin architecture to promote multiciliated cell formation

HIGHLIGHTS O_LIWDR5 has an H3K4 independent role in the formation of multiciliated cells.\nC_LIO_LIWDR5 controls apical cell expansion, basal body patterning, and ciliogenesis in multiciliated cells.\nC_LIO_LIWDR5 localizes near the ciliary base where it connects basal bodies to F-actin.\nC_LIO_LIWDR5 stabilizes the apical actin network in multiciliated cells.\nC_LI\n\nSUMMARYThe actin cytoskeleton is critical to shape cells and pattern intracellular organelles to drive tissue morphogenesis. In multiciliated cells (MCCs), apical actin forms a lattice that drives expansion of the cell surface necessary to host hundreds of cilia. The actin lattice also uniformly distributes basal bodies across this surface. This apical actin network is dynamically remodeled, but the molecules that regulate its architecture remain poorly understood. We identify the chromatin modifier, WDR5, as a regulator of apical F-actin in multiciliated cells. Unexpectedly, WDR5 functions independently of chromatin modification in MCCs. Instead, we discover a scaffolding role for WDR5 between the basal body and F-actin. Specifically, WDR5 binds to basal bodies and migrates apically, where F-actin organizes around WDR5. Using a monomer trap for G-actin, we show that WDR5 stabilizes F-actin to maintain apical lattice architecture. In summary, we identify a novel, non-chromatin role for WDR5 in stabilizing F-actin in multiciliated cells.\n\nIN BRIEFKulkarni et al discover a chromatin independent function for WDR5 in multiciliated cell formation. WDR5 localizes to the base of cilia and functions as a scaffold between the basal bodies and the apical actin lattice. There, WDR5 stabilizes the actin lattice that allows multiciliated cells to expand their apical surface, pattern basal bodies, and generate hundreds of cilia.

developmental biology↗

bioRxiv2017

RAPGEF5 regulates nuclear transport of β-catenin

Canonical Wnt signaling coordinates many critical aspects of embryonic development, while dysregulated Wnt signaling contributes to common diseases, including congenital malformations and cancer. The nuclear localization of {beta}-catenin is the defining step in pathway activation. However, despite intensive investigation, the mechanisms regulating {beta}-catenin nuclear transport remain undefined. In a patient with congenital heart disease and heterotaxy, a disorder of left-right patterning, we previously identified the guanine nucleotide exchange factor, RAPGEF5. Here, we demonstrate that RAPGEF5 regulates left-right patterning via Wnt signaling. In particular, RAPGEF5, regulates the nuclear translocation of {beta}-catenin independently of both {beta}-catenin cytoplasmic stabilization and the importin {beta}1/Ran mediated transport system. We propose a model whereby RAPGEF5 activates the nuclear GTPases, Rap1/2, to facilitate the nuclear transport of {beta}-catenin, defining a parallel nuclear transport pathway to Ran. Our results suggest new targets for modulating Wnt signaling in disease states.

developmental biology↗