Biology subjects

Valledor, M.

Publications and source records attributed to Valledor, M..

3 recordsLinked to original sources

bioRxiv2026

APP Dosage and Extracellular Domain Variants Drive Distinct Defects in Neurogenesis modeled in Down Syndrome iPS cells

The amyloid precursor protein (APP) is heavily studied as the source of amyloid beta in Alzheimers disease (AD), however, the complex functions of APP remain poorly understood, as does the impact of APP dosage on neurodevelopment. Here we study APP specifically in the context of Trisomy 21. In an effort to reduce APP dosage in trisomy 21 iPSCs, we generated trisomic isogenic lines which vary in APP dosage, including a full APP knock-out line, as well as lines carrying mutations of the APP extracellular domain. We used a panel of these lines to study potential impacts of APP dosage or structure on two distinct steps of neurogenesis in trisomic cells: 1) terminal differentiation of human neuro-progenitor cells (NPC) to post-mitotic neurons and 2) neuron structure as reflected in neurite outgrowth. Complete loss of APP causes marked defects in each of these two distinct steps, reducing both the terminal differentiation of NPCs to neurons, and proper neurite development for extended neuron structure. Hence, APP is necessary for both aspects of normal neurogenesis. Further analyses of the null and other mutant lines indicate that APP likely impacts these two distinct steps by two different mechanisms. Collective results suggest that the reduced terminal differentiation of NPCs reflects an effect of APP dosage, whereas the defects in neurite extension are due to structural mutation of the APP extracellular domain. Absence of APP or reduced (monosomic) APP dosage prolonged the cycling of trisomic NPCs, which is known to be regulated by Notch signaling. APP and Notch are the main targets of gamma-secretase cleavage, hence we hypothesized that APP dosage may impact neurogenesis indirectly, potentially via effects on Notch signaling. To test this, we treated NPCs with Compound E which inhibits gamma-secretase (and Notch signaling); results show this restored levels of neurogenesis in APP depleted lines, supporting an indirect effect of APP dosage. In contrast, results indicate that disruption of APP extracellular domain integrity impacts neurite extension via a more direct role of APP in neuron structural maturation. This study describes a resource of well-characterized APP mutant isogenic DS iPSC lines, implicates a dynamic interplay between APP dosage and Notch signaling, and raises new questions about the impact of APP dosage in orchestrating neural progenitor fate decisions during human brain development, specifically in the context of trisomy 21.

developmental biology↗

bioRxiv2022

Human XIST RNA acts early to condense architecture which facilitates A-repeat density-dependent initiation of gene silencing

XIST RNA triggers gene silencing chromosome-wide and transforms a euchromatic chromosome into a condensed Barr body. XIST is heavily studied in mouse ES cells, but here an inducible iPSC system allows analysis of initial steps in human chromosome silencing, revealing key points not known in either system. XIST RNA distribution was examined relative to biochemical and transcriptional changes directly within architecture of individual chromosome territories. Within a few hours of induction, XIST transcripts distribute as a large "sparse zone" and a smaller "dense zone", which, importantly, exhibit different effects on chromatin. Very sparse transcripts immediately trigger bright staining for H2AK119ub and CIZ1, a structural matrix protein. In contrast, H3K27me3 enrichment comes hours later and is much more restricted to the smaller dense RNA zone, which enlarges as the chromosome condenses. Importantly, silencing of several genes examined occurred well after architectural condensation, suggesting a possibly separable step. Surprisingly, we show the small A-repeat fragment of XIST can alone silence endogenous genes; however, results indicate this requires high local RNA density for effective histone deacetylation. Results support a concept whereby XIST RNA acts directly to condense the chromosome territory, comprised largely of non-coding DNA, which facilitates a required step to initiate gene silencing by the A-repeat. Hence, compacted architecture is not a consequence of collective gene silencing, but an early step required for chromosome-wide gene silencing.

cell biology↗

bioRxiv2018

Herpes ICP8 protein stimulates homologous recombination in human cells

Recombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red SynExo homologous recombination proteins Beta in Escherichia coli has approached 100% efficiency. While highly efficient in E. coli, recombineering using the Red SynExo in other organisms declines in efficiency roughly correlating with phylogenetic distance from E. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus Type 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstituted in vitro and shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a \"humanized\" version of Beta protein from phage {lambda}. ICP8 significantly enhanced gene targeting rates in HEK 293 T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.\n\nSIGNIFICANCEGenome modification by recombineering using SynExo viral recombination proteins has transformed functional genomic analysis in bacteria. Single-stranded DNA (ssDNA) recombineering approaches 100% efficiency in E. coli using Beta protein from bacteriophage lambda, but recombineering has not been extended to eukaryotic genomes. Efficient recombineering requires SynExos that co-evolved with a viral host, however SynExos are common to viruses infecting a variety of organisms, including humans. The ICP8 protein of Human Herpes virus Type 1 is a SynExo protein similar to Beta. In this pioneering study, Herpes ICP8 stimulated gene targeting in a human genome by homologous recombination while the bacterial virus Beta protein inhibited recombination in human cells. This is the first demonstration of host-specific recombineering in human cells using a human viral SynExo protein.

molecular biology↗