Biology subjects

Palmer, N.

Publications and source records attributed to Palmer, N..

7 recordsLinked to original sources

bioRxiv2024

Differential splice isoforms of mouse CDK2 play functionally redundant roles during mitotic and meiotic division

In most mammals, the cell cycle kinase; cyclin-dependent kinase 2 (CDK2) is expressed as two major isoforms due to the inclusion or exclusion of an alternatively spliced exon. The shorter CDK2 isoform: CDK2S, is expressed constitutively during the cell cycle and can be detected in many different tissue types. In contrast, the longer isoform: CDK2L, shows preferential expression in meiotically dividing cells of the germ cells and upon S-phase entry during mitotic cell division. Both CDK2L and CDK2S form heteromeric complexes with cyclins A2 and E1 in vitro. However, complexes comprised of each isoform differ considerably in their kinase activity towards known CDK substrates. It is currently unknown whether the long and short isoforms of CDK2 play functionally different roles in vivo during either mitotic and meiotic divisions as conventional knockout methodology leads to the loss of both isoforms. In this study, we find that both CDK2L and CDK2S are sufficient to support both mitotic and meiotic division when expressed in the absence of the other. This data contributes to the explanation of the apparent tolerance of the evolutionary loss of CDK2L expression in humans.

cell biology↗

bioRxiv2022

Extensive in vitro and in vivo protein translation via in situ circularized RNAs

RNAs are a powerful therapeutic class. However their inherent transience impacts their activity both as an interacting moiety as well as a template. Circularization of RNA has been demonstrated as a means to improve persistence, however simple and scalable approaches to achieve this are lacking. Utilizing autocatalytic RNA circularization, here we engineer in situ circularized RNAs (icRNAs). This approach enables icRNA delivery as simple linear RNA that is circularized upon delivery into the cell, thus making them compatible with routine synthesis, purification, and delivery formulations. We confirmed extensive protein translation from icRNAs both in vitro and in vivo and explored their utility in three contexts: first, we delivered the SARS-CoV-2 Omicron spike protein in vivo as icRNAs and showed corresponding induction of humoral immune responses; second, we demonstrated robust genome targeting via zinc finger nucleases delivered as icRNAs; and third, to enable compatibility between persistence of expression and immunogenicity, we developed a novel long range multiplexed (LORAX) protein engineering methodology to screen progressively deimmunized Cas9 proteins, and demonstrated efficient genome and epigenome targeting via their delivery as icRNAs. We anticipate this highly simple and scalable icRNA methodology could have broad utility in basic science and therapeutic applications.

bioengineering↗

bioRxiv2020

Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5 guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2 protein engineering via three approaches: First, we performed a novel deep mutational scan of the deaminase domain that enabled direct coupling of variants to corresponding RNA editing activity. Experimentally measuring the impact of every amino acid substitution across 261 residues, i.e. ~5000 variants, on RNA editing, revealed intrinsic domain properties, and also several mutations that greatly enhanced RNA editing. Second, we performed a domain-wide mutagenesis screen to identify variants that increased activity at 5-GA-3 motifs, and discovered novel mutants that enabled robust RNA editing. Third, we engineered the domain at the fragment level to create split deaminases. Notably, compared to full-length deaminase overexpression, split-deaminases resulted in >1000 fold more specific RNA editing. Taken together, we anticipate this comprehensive deaminase engineering will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

synthetic biology↗

bioRxiv2020

Population sequencing data reveal a compendium of mutational processes in human germline

Mechanistic processes underlying human germline mutations remain largely unknown. Variation in mutation rate and spectra along the genome is informative about the biological mechanisms. We statistically decompose this variation into separate processes using a blind source separation technique. The analysis of a large-scale whole genome sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. Seven of these processes lend themselves to a biological interpretation. One process is driven by bulky DNA lesions that resolve asymmetrically with respect to transcription and replication. Two processes independently track direction of replication fork and replication timing. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions. We also demonstrate that a recently discovered mutagenic process specific to oocytes can be localized solely from population sequencing data. This process is spread across all chromosomes and is highly asymmetric with respect to the direction of transcription, suggesting a major role of DNA damage.

genetics↗

bioRxiv2019

Inherited Causes of Clonal Hematopoiesis of Indeterminate Potential in TOPMed Whole Genomes

Age is the dominant risk factor for most chronic human diseases; yet the mechanisms by which aging confers this risk are largely unknown.1 Recently, the age-related acquisition of somatic mutations in regenerating hematopoietic stem cell populations was associated with both hematologic cancer incidence2-4 and coronary heart disease prevalence.5 Somatic mutations with leukemogenic potential may confer selective cellular advantages leading to clonal expansion, a phenomenon termed Clonal Hematopoiesis of Indeterminate Potential (CHIP).6 Simultaneous germline and somatic whole genome sequence analysis now provides the opportunity to identify root causes of CHIP. Here, we analyze high-coverage whole genome sequences from 97,691 participants of diverse ancestries in the NHLBI TOPMed program and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid, and inflammatory traits specific to different CHIP genes. Association of a genome-wide set of germline genetic variants identified three genetic loci associated with CHIP status, including one locus at TET2 that was African ancestry specific. In silico-informed in vitro evaluation of the TET2 germline locus identified a causal variant that disrupts a TET2 distal enhancer. Aggregates of rare germline loss-of-function variants in CHEK2, a DNA damage repair gene, predisposed to CHIP acquisition. Overall, we observe that germline genetic variation altering hematopoietic stem cell function and the fidelity of DNA-damage repair increase the likelihood of somatic mutations leading to CHIP.

genomics↗

bioRxiv2019

CDK2 kinase activity is a regulator of male germ cell fate

The ability of men to remain fertile throughout their lives depends upon establishment of a spermatogonial stem cell (SSC) pool from gonocyte progenitors, and also maintaining the proper balance between SSC renewal and spermatogenic differentiation throughout life. Depletion of SSCs causes infertility with a Sertoli Cell Only Syndrome (SCOS) phenotype. We previously created a mouse strain in which an inhibitory phosphorylation site (Tyr15) of Cyclin-dependent kinase 2 (Cdk2) was altered. Juvenile males homozygous for this allele (Cdk2Y15S) initiate the first round of spermatogenesis, which originates from prospermatogonia, but meiocytes arrest due to chromosomal defects resembling those in Cdk2-/- mice. Subsequent waves of spermatogonial differentiation and meiosis were largely absent, leading to an SCOS-like phenotype. Here, we demonstrate that Cdk2Y15S/Y15S mice possess mitotically active GFRa1+ SSC-like cells, but they are impaired in their ability to differentiate. Marker analysis and single cell RNA-seq revealed defective differentiation of gonocytes into SSCs. Biochemical and genetic data demonstrated that Cdk2Y15S is a gain-of-function allele causing deregulated kinase activity, and its phenotypic effects could be reversed by mutating the Thr160 positive regulatory site in cis. These results demonstrate that precise temporal regulation of CDK2 activity in male germ cell development and in the cell cycle is critical for long-term spermatogenic homeostasis.

genetics↗

bioRxiv2018

Exploring protein orthogonality in immune space: a case study with AAV and Cas9 orthologs

A major hurdle in protein-based therapeutics is the interaction with the adaptive immune system, which can lead to neutralization by circulating antibodies and clearance of treated cells by cytotoxic T-lymphocytes. One method of circumventing these issues is to use human or humanized proteins which avoid the immune response by self-recognition. However, this approach limits potential protein therapeutics to those of human origin, excluding many exciting effectors and delivery vehicles such as CRISPR-Cas9 and adeno-associated viruses (AAVs). To address this issue, we propose here the sequential use of orthologous proteins whose function is constrained by natural selection, but whose structure is subject to diversification by genetic drift. This would, in principle, allow for repeated treatments by immune orthogonal orthologs without reduced efficacy due to lack of immune cross-reactivity among the proteins. To explore and validate this concept we chose 91 Type II CRISPR-Cas9 orthologs and 167 AAV capsid protein orthologs, and developed a pipeline to compare total sequence similarity as well as predicted binding to class I and class II Major Histocompatibility Complex (MHC) proteins. Interestingly, MHC binding predictions revealed wide diversity among the set of Cas9 orthologs, with 83% of pairs predicted to have non cross-reacting immune responses, while no global immune orthogonality among AAV serotypes was observed. To confirm these findings we selected two Cas9 orthologs, from S. pyogenes and S. aureus, predicted to be orthogonal in immune space, and delivered them into mice via multiple AAV serotypes. We observed cross-reacting antibodies against AAV but not Cas9 orthologs in sera from immunized mice, validating the computationally predicted immune orthogonality among these proteins. Moving forward, we anticipate this framework can be applied to prescribe sequential regimens of immune orthogonal protein therapeutics to circumvent pre-existing or induced immunity, and eventually, to rationally engineer immune orthogonality among protein orthologs.

synthetic biology↗