Biology subjects

Douse, C. H.

Publications and source records attributed to Douse, C. H..

15 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

Retroviral insertions contributed to the divergence of human and chimpanzee brains

Over the past 5-7 million years, humans and chimpanzees have diverged in brain size, structural complexity, and cognitive abilities despite high conservation of protein-coding genes. Notably, the endogenization and proliferation of retroviral infections within host genomes has introduced numerous species-specific regulatory elements that have the potential to influence gene regulation. However, the role of these endogenous retroviruses in hominoid brain evolution remains unclear. A burst of lineage-specific PTERV1 retroviruses recently invaded the chimpanzee genome but are absent in humans. We conducted an epigenomic analysis of PTERV1 insertions in chimpanzee neural organoids and found that they are heavily covered by DNA methylation, representing more than 150 species-specific heterochromatin domains with the capacity to influence gene regulatory networks. We identified one such chimpanzee-specific PTERV1 insertion on chromosome 19 that blocks the expression of the long noncoding RNA LINC00662, via DNA methylation spread to the adjacent genomic region. The expression of LINC00662 was restored in chimpanzee induced pluripotent stem cells when we deleted the PTERV1 insertion using CRISPR editing. We found that LINC00662, a human-specific RNA, is highly expressed in the developing brain and plays an important role in the posttranscriptional control of neuronal maturation, axon outgrowth, and neural organoid development. In summary, our findings describe how endogenous retroviral insertions contributed to the functional divergence of the human and chimpanzee brains. This provides a new mechanism by which retroviral pandemics influenced primate brain speciation.

evolutionary biology↗

bioRxiv2025

A framework for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells

This protocol describes a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). It illustrates how to design gRNAs to target TE families or unique TE loci and how to validate the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach that combines bulk RNA sequencing, CUT&RUN epigenetic profiling, and proteomics. This unique framework optimizes performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models. For complete details on the use and execution of this protocol, please refer to Adami et al. (2025)1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/684366v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@154560aorg.highwire.dtl.DTLVardef@1dce192org.highwire.dtl.DTLVardef@5d7097org.highwire.dtl.DTLVardef@8aea84_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

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↗

bioRxiv2025

LINE-1 retrotransposons regulate the exit of human pluripotency and early brain development

Long interspersed nuclear element 1 (L1) retrotransposons represent a vast source of divergent genetic information. However, mechanistic analysis of whether and how L1s contribute to human developmental programs is lacking, in part due to the challenges associated with specific profiling and manipulation of human L1 expression. Here we show that thousands of hominoid-specific L1 integrants are expressed in human induced pluripotent stem cells and cerebral organoids. The activity of individual L1 promoters is surprisingly divergent and correlates with an active epigenetic state. Efficient on-target CRISPRi silencing of L1s revealed nearly a hundred co-opted L1-derived chimeric transcripts and L1 silencing resulted in changes in neural differentiation programs and reduced cerebral organoid size. Together, these data implicate L1s and L1-derived transcripts in hominoid-specific CNS developmental processes.

developmental 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

Pseudogenes limit the identification of novel common transcripts generated by their parent genes

The human genome contains numerous duplicated regions, such as parent-pseudogene pairs, causing sequencing reads to align equally well to either gene. The extent to which this ambiguity complicates transcriptomic analyses is currently unknown. This is concerning as many parent genes have been linked to disease, including GBA1, causally linked to both Parkinsons and Gaucher disease. We find that most of the short sequencing reads that map to GBA1, also map to its pseudogene, GBAP1. Using long-read RNA-sequencing in human brain, where all reads mapped uniquely, we demonstrate significant differences in expression compared to short-read data. We identify novel transcripts from both GBA1 and GBAP1, including protein-coding transcripts that are translated in vitro and detected in proteomic data, but that lack GCase activity. By combining long-read with single-nuclear RNA-sequencing to analyse brain-relevant cell types we demonstrate that transcript expression varies by brain region with cell-type-selectivity. Taken together, these results suggest a non-lysosomal function for both GBA1 and GBAP1 in brain. Finally, we demonstrate that inaccuracies in annotation are widespread among parent genes, with implications for many human diseases.

genetics↗

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↗

bioRxiv2020

A human-specific structural variation at the ZNF558 locus controls a gene regulatory network during forebrain development

The human forebrain has expanded in size and complexity compared to that of chimpanzee despite limited changes in protein-coding genes, suggesting that gene regulation is an important driver of brain evolution. Here we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate the mitophagy gene SPATA18, supporting a link between mitochondrial homeostasis and cortical expansion. The unusual on-off switch for ZNF558 expression resides in a downstream variable number tandem repeat (VNTR) that is contracted in humans relative to chimpanzee. Our data reveal the brain-specific co-option of a transposon-controlling KRAB-ZFP and how a human-specific regulatory network is established by a cis-acting structural genome variation. This represents a previously undescribed genetic mechanism in the evolution of the human brain.

genetics↗

bioRxiv2020

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

SummaryThe Human Silencing Hub (HUSH) complex epigenetically represses retroviruses, transposons and genes in vertebrates. HUSH therefore maintains genome integrity and is central in the interplay between intrinsic immunity, transposable elements and transcriptional regulation. Comprising three subunits - TASOR, MPP8 and Periphilin - HUSH regulates SETDB1-dependent deposition of the transcriptionally repressive epigenetic mark H3K9me3 and recruits MORC2 to modify local chromatin structure. However the mechanistic roles of each HUSH subunit remain undetermined. Here we show that TASOR lies at the heart of HUSH, providing a platform for assembling the other subunits. Targeted epigenomic profiling supports the model that TASOR binds and regulates H3K9me3 specifically over LINE-1 repeats and other repetitive exons in transcribed genes. We find TASOR associates with several components of the nuclear RNA processing machinery and its modular domain architecture bears striking similarities to that of Chp1, the central component of the yeast RNA-induced transcriptional silencing (RITS) complex. Together these observations suggest that an RNA intermediate may be important for HUSH activity. We identify the TASOR domains necessary for HUSH assembly and transgene repression. Structural and genomic analyses reveal that TASOR contains a poly-ADP ribose polymerase (PARP) domain dispensable for assembly and chromatin localization, but critical for epigenetic regulation of target elements. This domain contains a degenerated and obstructed active site and has hence lost catalytic activity. Together our data demonstrate that TASOR is a pseudo-PARP critical for HUSH complex assembly and H3K9me3 deposition over its genomic targets.

molecular biology↗

bioRxiv2019

Periphilin self-association underpins epigenetic silencing by the HUSH complex

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin forms -helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts.

biochemistry↗

bioRxiv2017

Neuropathic mutations in MORC2 perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms

Missense mutations in MORC2 cause neuropathies including spinal muscular atrophy and Charcot-Marie-Tooth disease. We recently identified MORC2 as an effector of epigenetic silencing by the HUSH complex. Here we report the biochemical and cellular activities of MORC2 variants, alongside crystal structures of wild-type and neuropathic forms of a human MORC2 fragment comprising the GHKL-type ATPase module and CW-type zinc finger. This fragment dimerizes upon binding ATP and contains a hinged, functionally critical coiled coil insertion absent in other GHKL ATPases. We find that dimerization and DNA binding of the MORC2 ATPase module transduce HUSH-dependent silencing. Disease mutations change the dynamics of dimerization by distinct structural mechanisms: destabilizing the ATPase-CW module, trapping the ATP lid or perturbing the dimer interface. These defects lead to modulation of HUSH function, thus providing a molecular basis for understanding MORC2-associated neuropathies.

biochemistry↗