Biology subjects

Alvarado, B.

Publications and source records attributed to Alvarado, B..

6 recordsLinked to original sources

bioRxiv2026

Engineered Allosteric Control Enhances Specificity and Potency of CRISPR-CasX-based Epigenetic Repressors

Epigenetic silencers are targeted therapeutics capable of persistently silencing genes without altering DNA sequence. However, many current silencers rely on direct fusion of a constitutively active DNA methyltransferase catalytic domain, that may cause off-target DNA methylation and cellular toxicity. In contrast, endogenous DNA methyltransferases contain a regulatory domain (ADD) that requires recognition of a permissive histone state before DNA methylation can occur. We reinstate this sequential proofreading logic in a compact CasX-based epigenetic repressor, generating Epigenetic Long-term CasX Repressors (ELXRs) that read local chromatin state before writing DNA methylation. In ELXRs, a repressor domain converts active chromatin into a state that unlocks the allosteric DNA methyltransferase. This creates a sequential, multi-gated process that preferentially restricts DNA methylation to the target site. Surprisingly, implementing this proofreading mechanism improved not only specificity, but also activity. ELXRs decreased off-target methylation up to 10-fold and rescued DNMT3A-dependent growth defects, with transcriptome profiling showing up to 150-fold fewer dysregulated genes, while also increasing on-target repression up to 4-fold across multiple loci. In mouse models, lipid nanoparticle delivery produced potent, durable PCSK9 silencing with precise promoter methylation. Together, these results demonstrate that coupling molecular recognition to effector activity through sequential proofreading can simultaneously improve specificity and potency.

molecular biology↗

bioRxiv2021

Molecular diversity and lineage commitment of human interneuron progenitors

Cortical interneurons are indispensable for proper function of neocortical circuits. Changes in interneuron development and function are implicated in human disorders, such as autism spectrum disorder and epilepsy. In order to understand human-specific features of cortical development as well as the origins of neurodevelopmental disorders it is crucial to identify the molecular programs underlying human interneuron development and subtype specification. Recent studies have explored gene expression programs underlying mouse interneuron specification and maturation. We applied single-cell RNA sequencing to samples of second trimester human ganglionic eminence and developing cortex to identify molecularly defined subtypes of human interneuron progenitors and immature interneurons. In addition, we integrated this data from the developing human ganglionic eminences and neocortex with single-nucleus RNA-seq of adult cortical interneurons in order to elucidate dynamic molecular changes associated with commitment of progenitors and immature interneurons to mature interneuron subtypes. By comparing our data with published mouse single-cell genomic data, we discover a number of divergent gene expression programs that distinguish human interneuron progenitors from mouse. Moreover, we find that a number of transcription factors expressed during prenatal development become restricted to adult interneuron subtypes in the human but not the mouse, and these adult interneurons express species- and lineage-specific cell adhesion and synaptic genes. Therefore, our study highlights that despite the similarity of main principles of cortical interneuron development and lineage commitment between mouse and human, human interneuron genesis and subtype specification is guided by species-specific gene programs, contributing to human-specific features of cortical inhibitory interneurons.

neuroscience↗

bioRxiv2018

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution

Direct comparisons of human and non-human primate brain tissue have the potential to reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is largely inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. First, we systematically evaluated the fidelity of organoid models to primary human and macaque cortex, finding organoid models preserve gene regulatory networks related to cell types and developmental processes but exhibit increased metabolic stress. Second, we identified 261 genes differentially expressed in human compared to chimpanzee organoids and macaque cortex. Many of these genes overlap with human-specific segmental duplications and a subset suggest increased PI3K/AKT/mTOR activation in human outer radial glia. Together, our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

developmental biology↗

bioRxiv2018

Regulation of Cell-Type-Specific Transcriptomes by miRNA Networks During Human Brain Development

MicroRNAs (miRNAs) regulate many cellular events by regulating hundreds of mRNA transcripts. However, it is unclear how miRNA-mRNA interactions are contextualized into the framework of transcriptional heterogeneity among closely related cells of the developing human brain. By combining the multiple complementary approaches, AGO2-HITS-CLIP, single-cell profiling and bipartite network analysis, we show that the miRNA-mRNA network operates as functional modules related to cell-type identities and undergo dynamic transitions during brain development.

developmental biology↗

bioRxiv2018

A draft single-cell atlas of human glioblastoma reveals a single axis of phenotype in tumor-propagating cells.

Tumor-propagating glioblastoma (GBM) stem-like cells (GSCs) of the proneural and mesenchymal molecular subtypes have been described. However, it is unknown if these two GSC populations are sufficient to generate the spectrum of cellular heterogeneity observed in GBM. The lineage relationships and niche interactions of GSCs have not been fully elucidated. We perform single-cell RNA-sequencing (scRNA-seq) and matched exome sequencing of human GBMs (12 patients; >37,000 cells) to identify recurrent hierarchies of GSCs and their progeny. We map sequenced cells to tumor-anatomical structures and identify microenvironment interactions using reference atlases and quantitative immunohistochemistry. We find that all GSCs can be described by a single axis of variation, ranging from proneural to mesenchymal. Increasing mesenchymal GSC (mGSC) content, but not proneural GSC (pGSC) content, correlates with significantly inferior survival. All clonal expressed mutations are found in the GSC populations, with a greater representation of mutations found in mGSCs. While pGSCs upregulate markers of cell-cycle progression, mGSCs are largely quiescent and overexpress cytokines mediating the chemotaxis of myeloid-derived suppressor cells. We find mGSCs enriched in hypoxic regions while pGSCs are enriched in the tumors invasive edge. We show that varying proportions of mGSCs, pGSCs, their progeny and stromal/immune cells are sufficient to explain the genetic and phenotypic heterogeneity observed in GBM. This study sheds light on a long-standing debate regarding the lineage relationships between GSCs and other glioma cell types.

cancer biology↗

bioRxiv2018

Massively parallel dissection of human accelerated regions in human and chimpanzee neural progenitors

Using machine learning (ML), we interrogated the function of all human-chimpanzee variants in 2,645 Human Accelerated Regions (HARs), some of the fastest evolving regions of the human genome. We predicted that 43% of HARs have variants with large opposing effects on chromatin state and 14% on neurodevelopmental enhancer activity. This pattern, consistent with compensatory evolution, was confirmed using massively parallel reporter assays in human and chimpanzee neural progenitor cells. The species-specific enhancer activity of assayed HARs was accurately predicted from the presence and absence of transcription factor footprints in each species. Despite these striking cis effects, activity of a given HAR sequence was nearly identical in human and chimpanzee cells. These findings suggest that HARs did not evolve to compensate for changes in the trans environment but instead altered their ability to bind factors present in both species. Thus, ML prioritized variants with functional effects on human neurodevelopment and revealed an unexpected reason why HARs may have evolved so rapidly.

evolutionary biology↗