Biology subjects

Pandiloski, N.

Publications and source records attributed to Pandiloski, N..

7 recordsLinked to original sources

bioRxiv2026

De novo missense variants in TRIM28 identified in individuals with neurodevelopmental delay show features of transposable element activation

TRIM28 is an epigenetic co-repressor protein that silences transposable elements (TEs). Although loss-of-function studies in mice led to neurodevelopmental defects, a functional link between TRIM28 and human neurodevelopment has yet to be established. In this study, we describe two patients with neurodevelopmental delay who carry de novo TRIM28 missense variants. Using CRISPR-edited induced pluripotent stem cell lines and differentiated neural organoids, we demonstrate that these variants result in the loss of the histone mark H3K9me3 over TEs. This releases the regulatory potential of TEs resulting in altered expression of nearby genes. These findings could be replicated using CRISPRi-based TRIM28 silencing, which suggests that the two variants result in a loss of function. Our results highlight the critical role of TRIM28 in regulating TEs during human brain development, establishing a link between TRIM28 variants and neurodevelopmental delay. One Sentence SummaryTRIM28 variants disrupt epigenetic control of transposable elements in the developing human brain, linking them to neurodevelopmental delay.

neuroscience↗

bioRxiv2025

MORC2 directs transcription-dependent CpG methylation of human LINE-1 transposons in early neurodevelopment

Methylation of CpG dinucleotides is essential for silencing genomic repeats such as LINE-1 retrotransposons (L1s) in the germline and soma. Evolutionarily-young L1s are transcribed in human pluripotent stem cells, but how CpG methylation is patterned to these L1s upon exit of pluripotency is unknown. Here we investigate the critical functions of chromatin regulator MORC2 in epigenome reprogramming of the repetitive genome in early human neurodevelopment. We find that reversible ATP-dependent dimerization is required for MORC2 accumulation over L1s but not gene promoters. Engineered mutations of the MORC2 ATPase module severely disrupts the distribution of MORC2 chromatin binding, leading to simultaneous loss of L1 transcriptional control and hyper-repression of clustered ZNF genes in human pluripotent stem cells. Upon neural differentiation these phenotypes persist due to striking, targeted defects in CpG methylation patterning. Together our results define the vital role of MORC2 in safeguarding the somatic human genome upon exit of pluripotency by directing CpG methylation patterning over transcriptionally-active retrotransposons in a manner analogous to the piRNA pathway in the germline.

molecular biology↗

bioRxiv2025

Loss of H3K9me3 maintenance in human neural progenitor cells leads to transcriptional activation of L1 retrotransposons

Heterochromatin is characterised by an inaccessibility to the transcriptional machinery and is associated with the histone mark H3K9me3. However, studying the functional consequences of heterochromatin loss in human cells has been challenging. Here, we used CRISPRi-mediated silencing of the histone methyltransferase SETDB1 to remove H3K9me3 heterochromatin in human neural progenitor cells. Despite a major loss of H3K9me3 peaks resulting in genome-wide reorganization of heterochromatin domains, silencing of SETDB1 had a limited effect on cell viability. Cells remained proliferative and expressed appropriate marker genes. We found that a key event following the loss of SETDB1-mediated H3K9me3 was the expression of evolutionarily young L1 retrotransposons. Derepression of L1s was associated with a loss of CpG DNA methylation at their promoters, suggesting that deposition of H3K9me3 at the L1 promoter is required to maintain DNA methylation. In conclusion, these results demonstrate that loss of H3K9me3 in human neural somatic cells transcriptionally activates evolutionary young L1 retrotransposons.

molecular biology↗

bioRxiv2023

Mini-heterochromatin domains constrain the cis-regulatory impact of SVA transposons in human brain development and disease

SVA retrotransposons remain active in humans and contribute to individual genetic variation. Polymorphic SVA alleles harbor gene-regulatory potential and can cause genetic disease. However, how SVA insertions are controlled and functionally impact human disease is unknown. Here, we dissect the epigenetic regulation and influence of SVAs in cellular models of X-linked dystonia-parkinsonism (XDP), a neurodegenerative disorder caused by an SVA insertion at the TAF1 locus. We demonstrate that the KRAB zinc finger protein ZNF91 establishes H3K9me3 and DNA methylation over SVAs, including polymorphic alleles, in human neural progenitor cells. The resulting mini-heterochromatin domains attenuate the cis-regulatory impact of SVAs. This is critical for XDP pathology; removal of local heterochromatin severely aggravates the XDP molecular phenotype, resulting in increased TAF1 intron retention and reduced expression. Our results provide unique mechanistic insights into how human polymorphic transposon insertions are recognized, and their regulatory impact constrained by an innate epigenetic defense system.

neuroscience↗

bioRxiv2023

DNA methylation governs the sensitivity of repeats to restriction by the HUSH-MORC2 corepressor

The human silencing hub (HUSH) complex binds to transcripts of LINE-1 retrotransposons (L1s) and other genomic repeats, recruiting MORC2 and other effectors to remodel chromatin. However, how HUSH and MORC2 operate alongside DNA methylation, a central epigenetic regulator of repeat transcription, remains poorly understood. Here we interrogate this relationship in human neural progenitor cells (hNPCs), a somatic model of brain development that tolerates removal of DNA methyltransferase DNMT1. Upon loss of MORC2 or HUSH subunit TASOR in hNPCs, L1s remain silenced by robust promoter methylation. However, genome demethylation and activation of evolutionarily-young L1s attracts MORC2 binding. Simultaneous depletion of DNMT1 and MORC2 causes massive accumulation of L1 transcripts. We identify the same mechanistic hierarchy at pericentromeric -satellites and clustered protocadherin genes, repetitive elements important for chromosome structure and neurodevelopment respectively. Our data delineate the independent epigenetic control of repeats in somatic cells, with implications for understanding the vital functions of HUSH-MORC2 in hypomethylated contexts throughout human development.

molecular biology↗

bioRxiv2023

L1 retrotransposons drive human neuronal transcriptome complexity and functional diversification

The genetic mechanisms underlying the expansion in size and complexity of the human brain remains poorly understood. L1 retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution is largely unknown. Using multi-omic profiling we here demonstrate that L1-promoters are dynamically active in the developing and adult human brain. L1s generate hundreds of developmentally regulated and cell-type specific transcripts, many which are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived lncRNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPRi-silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

neuroscience↗

bioRxiv2022

Structure and functional mapping of the KRAB-KAP1 repressor complex

Transposable elements (TEs) are a genetic reservoir from which new genes and regulatory elements can emerge. Expression of TEs can be pathogenic, however, and is tightly regulated. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) recruit co-repressor KRAB-associated protein 1 (KAP1/TRIM28) to regulate many TEs but how KRAB-ZFPs and KAP1 interact remains unclear. Here, we report the crystal structure of the KAP1 tripartite motif (TRIM) in complex with the KRAB domain from a human KRAB-ZFP, ZNF93. Structure-guided mutations in the KAP1-KRAB binding interface abolished repressive activity in an epigenetic TE silencing assay. Deposition of H3K9me3 over thousands of loci is lost genome-wide in cells expressing a KAP1 variant with mutations that abolish KRAB binding. Our work identifies and functionally validates the KRAB-KAP1 molecular interface, which forms the nexus of a transcriptional control axis vital to vertebrates and underpins programmable gene repression by CRISPRi.

biochemistry↗