Biology subjects

da Rocha, S. T.

Publications and source records attributed to da Rocha, S. T..

6 recordsLinked to original sources

bioRxiv2025

Stem-cell modeling of cerebellar dysfunction of Angelman syndrome

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function of the maternal allele of the neuron-specific imprinted gene UBE3A. Although AS presents with developmental delay, ataxia, epilepsy, and speech impairment, the distinct roles of various brain regions in its pathology remain unclear, hindering the design of effective therapeutic strategies. Of all brain regions, the nature and impact of dysfunction of the cerebellum in AS remain elusive. To start addressing this, we leveraged our optimized stem cell-based model of regionally patterned human cerebellar organoids (hCerOs) capable of producing major cerebellar neuronal subtypes, including Purkinje and granule cells, further matured in a two-dimensional (2D) culture system. We modeled AS using induced pluripotent stem cell (iPSC) lines carrying the common class II deletion and unaffected controls previously validated in our system. hCerOs from both backgrounds recapitulate lineage commitment and neuron-specific UBE3A imprinting. AS hCerOs consistently show reduced size, impaired neuroepithelial expansion, and diminished expression of glutamatergic and GABAergic progenitor markers. Transcriptomic profiling and functional assays assessing neuronal activity during the maturation stage uncovered delayed maturation, abnormal firing and increased neuronal excitability in AS cultures. These results position our hCerO model as a robust platform for future mechanistic studies and therapeutic screening aimed at targeting cerebellar dysfunction in AS.

developmental biology↗

bioRxiv2024

Erosion of X-Chromosome Inactivation in female hiPSCs is heterogeneous and persists during differentiation

During culture, female human pluripotent stem cells (hPSCs), including human induced PSCs (hiPSCs) exhibit a propensity for erosion of X-chromosome inactivation (XCI). This phenomenon is characterized by the loss of XIST RNA expression and reactivation of a subset of X-linked genes from the inactive X chromosome (Xi). XCI erosion, despite its common occurrence, is often overlooked by the stem cell community, hindering a complete understanding of its impact on both fundamental and translational applications of hiPSCs. Investigating erosion dynamics in female hiPSCs, our study reveals that XCI erosion is a frequent yet heterogeneous phenomenon, resulting in the reactivation of several X-linked genes. The likelihood of a gene to erode increases for those located on the short arm of the X chromosome and within H3K27me3-enriched domains. Paradoxically, genes that typically escape XCI are hypersensitive to loss of XIST RNA and XCI erosion. This implies that XIST RNA normally restrains expression levels of these genes on the Xi. Importantly, increased X-linked gene expression upon erosion does not globally impact (hydroxy)methylation levels in hiPSCs or at imprinted regions. By exploring diverse differentiation paradigms, such as trilineage commitment and cardiac differentiation, our study reveals the persistence of abnormal XCI patterns throughout differentiation. This finding has significant implications for fundamental research, translational applications, and clinical use of stem cells. We underscore the importance of raising awareness within the stem cell community regarding XCI erosion and advocate for its inclusion in comprehensive hiPSC quality control.

molecular biology↗

bioRxiv2023

Stability of Genomic Imprinting and X-Chromosome Inactivation in the Aging Brain

Epigenetic drift is a hallmark of aging that contributes to the irreversible decline in organismal fitness ultimately leading to aging-related diseases. Epigenetic modifications regulate the cellular memory of the epigenetic processes of genomic imprinting and X-chromosome inactivation to ensure monoallelic expression of imprinted and X-linked genes. Whether epigenetic drift affects maintenance of genomic imprinting and X-chromosome inactivation has not been comprehensively studied. Here, we investigate the allele-specific transcriptional and epigenetic signatures of the aging brain, by comparing juvenile and old hybrid mice obtained from C57BL/6J (BL6) & CAST/EiJ (CAST) reciprocal crosses, with an emphasis on the hippocampus (HCP). We confirm that the aged HCP shows an increase of DNA hydroxymethylation, a sign of epigenetic drift, and a typical aging transcriptional signature. Genomic imprinting was found to be largely unaffected with stable parent-of-origin-specific DNA methylation in HCP, but also other brain regions such as the cerebellum (CB), nucleus accumbens, hypothalamus and prefrontal cortex. Consistently, transcriptomics analysis confirmed unaltered imprinting expression in the aged HCP. An exception are three novel non-coding transcripts (B230209E15Rik, Ube2nl and A330076H08Rik) at the Prader-Willi syndrome/Angelman syndrome (PWS/AS) imprinted locus which lose strict monoallelic expression during aging. Like imprinting, X-chromosome inactivation was remarkably stable with no signs of aging-driven skewing or relaxation of monoallelic expression of X-linked genes. Our study provides a valuable resource for evaluating monoallelic expression in the aging brain and reveals that, despite epigenetic drift during aging, genomic imprinting and X-chromosome inactivation remain predominantly stable throughout the process of physiological aging in the mouse brain.

molecular biology↗

bioRxiv2020

Tracking H3K27me3 and H4K20me1 dynamics during XCI reveals similarities in recruitment mechanism

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long-noncoding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the molecular mechanism allowing for H4K20me1 enrichment. To follow XCI dynamics in living cells, we developed a genetically-encoded, H3K27me3-specific intracellular antibody, or H3K27me3-mintbody. By combining it with live-imaging of H4K20me1, the X chromosome and Xist RNA we uncover similarities in the initial accumulation dynamics of H3K27me3 and H4K20me1. Further ChIP-seq analysis confirmed concurrent accumulation of both marks during XCI albeit with distinct genomic distributions. Using a Xist B and C repeat mutant, which can silence the X but does not allow for H3K27me3 deposition, we also found a lack of H4K20me1 enrichment. Thus, these two marks accumulate at the X thanks to the same region of Xist and H4K20me1 in particular may have a role in the chromatin compaction that characterises facultative heterochromatin.

developmental biology↗

bioRxiv2020

IMPLICON: an ultra-deep sequencing method to uncover DNA methylation at imprinted regions

Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a new high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We initially designed IMPLICON to look at ICRs in adult tissues of inbred mice. Then, we validated it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.

molecular biology↗

bioRxiv2018

Exploring the role of Polycomb recruitment in Xist-mediated silencing of the X chromosome in ES cells

Xist RNA has been established as the master regulator of X-chromosome inactivation (XCI) in female eutherian mammals but its mechanism of action remains unclear. By creating novel Xist mutants at the endogenous locus in mouse embryonic stem (ES) cells, we dissect the role of the conserved A-B-C-F repeats. We find that transcriptional silencing can be largely uncoupled from Polycomb repressive complex 1 and 2 (PRC1/2) recruitment, which requires repeats B and C. Xist {Delta}B+C RNA specifically loses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners is largely unaffected. However, a slight relaxation of transcriptional silencing in Xist {Delta}B+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways. In this context, Polycomb recruitment seems to be of moderate relevance in the initiation of silencing.

genetics↗