Biology subjects

Marzi, S. J.

Publications and source records attributed to Marzi, S. J..

12 recordsLinked to original sources

bioRxiv2026

Best Practices for Pre-Processing Large-Scale ATAC-Seq Data: Why Epidemiological Studies Demand Greater Stringency

Assay for transposase accessible chromatin using sequencing (ATAC-seq) and related techniques provide critical insight into the epigenetic mechanisms that regulate gene expression in health and disease. Accurate identification of biologically meaningful chromatin accessibility signals remains an underexplored analytical challenge. Advances in sequencing technologies have increased both the magnitude and complexity of ATAC-seq datasets, leading to demand for scalable, reproducible and robust computational analysis frameworks. Peak calling, which identifies regions of chromatin accessibility, is a critical step that underpins all downstream quantitative analyses. However, there is a lack of consensus regarding the optimal peak calling strategy. Here, we present a novel bioinformatics pipeline PIPPA (Pipeline for Integrative Peak calling in Population-scale ATAC-seq), for quality control and peak set curation of large-scale ATAC-seq datasets. Using two independent cell-type sorted ATAC-seq datasets, we show that peak calling performance is dataset-dependent and that no single significance threshold performs optimally across all scenarios. We provide practical recommendations for parameters to generate high-confidence chromatin accessibility profiles. Our peak calling approach is applicable for other genome wide epigenomic assays such as ChIP-seq and CUT&Tag. By standardising this process, the PIPPA pipeline enhances epidemiological studies focused in identifying epigenetic dysregulation associated with development and disease.

bioinformatics↗

bioRxiv2026

Cell type-specific histone acetylation landscape in Alzheimer's disease reveals a putative role of MITF in microglia

Alzheimer's disease (AD) is characterised by aberrant amyloid beta and tau aggregation, neuroinflammation, demyelination and neurodegeneration, which have been linked to changes in cell-specific gene expression signatures. Among the mechanisms driving cell-type-specific transcriptional changes, histone acetylation plays a central role in regulating gene activity. While global alterations in histone acetylation have been implicated in AD, the contribution of individual cell types to these epigenetic changes remains poorly understood. To decode cell-type-specific changes in gene regulation in AD pathogenesis, we profiled histone H3 lysine 27 acetylation (H3K27ac) in microglia, oligodendrocytes and neurons from the prefrontal cortex of individuals with late-stage AD and non-dementia controls. Oligodendrocytes had the highest number of differential H3K27ac regions in AD, followed by microglia. Genes nearest to differential H3K27ac in purified microglia were enriched for phagocytosis, lipid processing, inflammatory and disease-associated cell state signature genes. Gene network analysis revealed downregulation of homeostatic genes in AD microglia and upregulation of immune activation, including signatures of lipid-handling and monocyte-derived macrophages. Oligodendrocyte co-regulated regions were indicative of increased MHC class I antigen presentation and altered neuron-oligodendrocyte interactions in AD. We identified H3K27ac allele-specific variants (ASVs) enriched near endolysosomal and ubiquitin-proteasome-associated genes in microglia and neurons. ASVs coincided with AD genome-wide association study (GWAS) risk loci, including CLU in oligodendrocytes and HLA-DRB1 in microglia. DNA motif analysis identified putative transcription factor drivers of AD glial dysregulation, including the lysosomal-associated MITF, Cap'n'collar (CNC) family (BACH1 and NFE2) and AP-1 activation in microglia. DNA binding of the MITF protein in human microglia was localised to lysosomal-associated genes and enriched in H3K27ac regions upregulated in AD and near disease-associated microglia (DAM) genes. Collectively, these findings implicate lysosomal dysfunction and upstream transcriptional regulation via MITF as key processes in AD microglia.

neuroscience↗

bioRxiv2025

Optimised fluorescence-activated nuclei sorting for epigenomic analysis of cortical cell types

Increased understanding of the functional complexity of the genome has led to growing recognition of the role of non-sequence-based regulatory variation in disorders of the human central nervous system. Most genomic analyses of the brain are limited by the use of bulk tissue, which comprises a heterogeneous mix of different neural cell types with distinct epigenetic profiles, thereby limiting the ability to attribute regulatory changes to specific cell populations. Given the limited availability of human post-mortem tissue resources and the importance of integrating multi-omic data from the same samples, there is a critical need for methods that enable parallel, cell-type-resolved genomic profiling. We present optimised protocols using fluorescence-activated nuclei sorting (FANS) to isolate nuclei from different human and mouse brain cell types for downstream multi-omic analysis. Our approach enables the robust purification of neuronal, oligodendrocyte, microglial and other glial-origin nuclei from both adult and fetal brain tissue. We demonstrate that FANS-isolated nuclei are compatible with a wide range of genomic assays, including profiling of DNA modifications, histone modifications, chromatin accessibility, and gene expression. This protocol maximises the utility of limited post-mortem tissue resources and provides a unified workflow for comprehensive, cell-type-specific interrogation of molecular mechanisms involved in the brain.

genomics↗

bioRxiv2025

17q21.31 locus regulates Parkinson's disease relevant pathways through KANSL1 activity

An inversion polymorphism at the 17q21.31 locus defines the H1 and H2 haplotypes, with the former linked to multiple neurodegenerative disorders, including an increased risk of Parkinsons disease (PD). Although the high linkage disequilibrium at this locus has made it difficult to decipher which gene(s) drive the PD association, there is increasing evidence to support the role of KANSL1 as a risk gene. KANSL1 has been shown to regulate the expression of some PD-associated genes and pathways, likely as part of the histone acetylating non-specific lethal (NSL) complex. Here for the first time, we studied the global effects of 17q21.31 haplotype variation using bulk and single-nuclear RNA-sequencing data from control and PD patient brain. We first analysed differential gene expression across haplotype groups, and then assessed the contribution of KANSL1 by comparing with the results of an siRNA knockdown in neuronal and glial human cell lines. We demonstrated that the PD risk-associated H1 haplotype downregulates autophagy, lysosomal and mitochondrial processes, all of which have already been implicated in PD aetiology. Furthermore, these effects were apparent in both neuronal and glial cell types, and in the case of the latter, appear to be associated with the modulation of innate and adaptive immune responses. Thus, we identify important links between NSL complex activity and PD pathophysiology that can be leveraged for novel therapeutic interventions.

neuroscience↗

bioRxiv2024

Unique nigral and cortical pathways implicated by epigenomic and transcriptional analyses in a rotenone rat model of Parkinson's disease

Pesticide exposure is increasingly recognised as a potential environmental factor contributing to the onset of idiopathic Parkinsons disease, yet the molecular mechanisms underlying this connection remain unclear. This study aims to explore how pesticide exposure disrupts key brain regions involved in Parkinsons disease pathology by reshaping gene regulatory landscapes. Using the well-established rotenone rat model of the disease, we performed H3K27ac ChIP-sequencing to profile active regulatory elements in the substantia nigra and cortex. In this model, rotenone distributes uniformly throughout the brain, and the degree of complex I inhibition is equivalent in cortical and substantia nigra neurons. Despite the uniformity of complex I inhibition, we identified widespread epigenomic differences, with brain region specific acetylation patterns associated with rotenone exposure. We showed consistent changes in transcriptomic activity by RNA-sequencing. Our results indicate there is a strong immune response to rotenone localised to the substantia nigra and highlight an enrichment of immune-related motifs in this brain region, suggesting that the immune response is at least partially driven by gene regulatory mechanisms. We also noted an increase in C1q complement pathway activity in the substantia nigra. In contrast, we identified widespread dysregulation of synaptic function at the gene regulatory level in the cortex of these same rats. Our results highlight a role for gene regulatory mechanisms potentially mediating the effects of pesticide exposure, driving region-specific functional responses in the brain that may contribute to the pathology and selective vulnerability that characterise Parkinsons disease.

genomics↗

bioRxiv2024

Generation of morphologically distinct astrocyte subtypes from human iPSCs reveals a link between glial shape, function and reactivity

Astrocyte morphology in vivo is heterogeneous across different subtypes and dynamically changes in response to various stimuli. However, several questions on the mechanistic links between shape and function remain unanswered. Here, we developed an efficient protocol to generate pure populations of morphologically distinct human astrocytes in vitro, which we used for a systematic analysis of shape-function relationships. We performed a structural, molecular, and functional characterization of these populations and highlighted how their distinct morphologies mirror distinct functional and transcriptional patterns at the population level. We were also able to both correlate gene expression profiles of these morphologically distinct astrocyte subtypes with in vivo astrocytes in the human brain, and to validate our findings with primary isolated murine astrocytes in vitro. Moreover, we show that the observed morphological differences are correlated with changes in key cytoskeletal proteins, which offers a potential link to the observed functional differences. Finally, we demonstrated that different morphological subtypes of astrocytes have distinct reactivity responses to a common stimulus. This study offers a glimpse into the shape-function dynamics of human astrocytes, highlighting potential mechanistic links between cytoskeletal usage and astrocyte function, while also providing tools and datasets that will be useful for further studies into human glial biology in health and disease.

neuroscience↗

bioRxiv2024

Genetic risk for neurodegenerative conditions is linked to disease-specific microglial pathways

Genome-wide association studies have identified thousands of common variants associated with an increased risk of neurodegenerative disorders. However, the noncoding localization of these variants has made the assignment of target genes for brain cell types challenging. Genomic approaches that infer chromosomal 3D architecture can link noncoding risk variants and distal gene regulatory elements such as enhancers to gene promoters. By using enhancer-to-promoter interactome maps for microglia, neurons, and oligodendrocytes, we identified cell-type-specific enrichment of genetic heritability for brain disorders through stratified linkage disequilibrium score regression. Our analysis suggests that genetic heritability for multiple neurodegenerative disorders is enriched at microglial chromatin contact sites. Through Hi-C coupled multimarker analysis of genomic annotation (H-MAGMA) we identified disease risk genes for Alzheimers disease, Parkinsons disease, multiple sclerosis and amyotrophic lateral sclerosis. We found that disease-risk genes were overrepresented in microglia compared to other brain cell types across neurodegenerative conditions. Notably, the microglial risk genes and pathways identified were largely specific to each disease. Our findings reinforce microglia as an important, genetically informed cell type for therapeutic interventions in neurodegenerative conditions and highlight potentially targetable disease-relevant pathways.

genomics↗

bioRxiv2024

The APOE isoforms differentially shape the transcriptomic and epigenomic landscapes of human microglia in a xenotransplantation model of Alzheimer's disease

Microglia play a key role in the response to amyloid beta in Alzheimers disease (AD). In this context, a major transcriptional response of microglia is the upregulation of APOE, the strongest late-onset AD risk gene. Of its three isoforms, APOE2 is thought to be protective, while APOE4 increases AD risk. We hypothesised that the isoforms functionally alter microglia by shaping their transcriptomic and chromatin landscapes. We used RNA- and ATAC-sequencing to profile gene expression and chromatin accessibility of human microglia isolated from a xenotransplantation model of AD. We identified widespread transcriptomic and epigenomic differences which are dependent on APOE genotype, and are corroborated across the profiling assays. Our results indicate that impaired microglial proliferation, migration and immune responses may contribute to the increased risk for late-onset AD in APOE4 carriers, while increased DNA-binding of the vitamin D receptor in APOE2 microglia may contribute to the isoforms protective role.

genomics↗

bioRxiv2024

Predicting gene expression from histone marks using chromatin deep learning models depends on histone mark function, regulatory distance and cellular states

To understand the complex relationship between histone mark activity and gene expression, recent advances have used in silico predictions based on large-scale machine learning models. However, these approaches have omitted key contributing factors like cell state, histone mark function or distal effects, that impact the relationship, limiting their findings. Moreover, downstream use of these models for new biological insight is lacking. Here, we present the most comprehensive study of this relationship to date - investigating seven histone marks, in eleven cell types, across a diverse range of cell states. We used convolutional and attention-based models to predict transcription from histone mark activity at promoters and distal regulatory elements. Our work shows that histone mark function, genomic distance and cellular states collectively influence a histone marks relationship with transcription. We found that no individual histone mark is consistently the strongest predictor of gene expression across all genomic and cellular contexts. This highlights the need to consider all three factors when determining the effect of histone mark activity on transcriptional state. Furthermore, we conducted in silico histone mark perturbation assays, uncovering functional and disease related loci and highlighting frameworks for the use of chromatin deep learning models to uncover new biological insight. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/587323v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@18dfcbcorg.highwire.dtl.DTLVardef@cba6b8org.highwire.dtl.DTLVardef@2eb0ceorg.highwire.dtl.DTLVardef@159d5b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2022

CUT&Tag recovers up to half of ENCODE ChIP-seq peaks

Techniques for genome-wide epigenetic profiling have been undergoing accelerated development toward recovery of high-quality data from bulk and single cell samples. DNA-protein interactions have traditionally been profiled via chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq), which has become the gold standard for studying histone modifications or transcription factor binding. Cleavage Under Targets & Tagmentation (CUT&Tag) is a rapidly expanding new technique that enables profiling of such interactions in situ at high sensitivity and is adaptable to single cell applications. However, thorough evaluation and benchmarking against established ChIP-seq datasets are lacking. Here, we comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Combining multiple new and published CUT&Tag datasets, there was an average recall of 54% known ENCODE peaks for both histone modifications. To optimize data analysis steps, we tested peak callers MACS2 and SEACR and identified optimal peak calling parameters. Considering both precision and recall of known ENCODE peaks, the peak callers were comparable in their performance, although peaks produced by MACS2 match ENCODE peak width distributions more closely. We found that reducing PCR cycles during library preparation lowered duplication rates at the expense of ENCODE peak recovery. Despite the moderate ENCODE peak recovery, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments, providing a benchmarking framework for the experimental design and analysis of CUT&Tag studies, and will facilitate future efforts to apply CUT&Tag in human tissues and single cells.

genomics↗

bioRxiv2021

CHAS, a deconvolution tool, infers cell type-specific signatures in bulk brain histone acetylation studies of brain disorders

Acetylation of histone H3 lysine 27 (H3K27ac) has emerged as an informative disease-associated epigenetic mark. However, cell type-specific contributions to epigenetic dysregulation in disease are unclear as studies have often used bulk brain tissue. Therefore, methods for the deconvolution of bulk H3K27ac profiles are critical. Here we developed the Cell type-specific Histone Acetylation Score (CHAS), a computational tool for inferring cell type-specific signatures in bulk brain H3K27ac profiles. We applied CHAS to > 300 H3K27ac ChIP-seq samples from studies of Alzheimers disease, Parkinsons disease, autism spectrum disorder, schizophrenia, and bipolar disorder in bulk post-mortem brain tissue. In addition to recapitulating known disease-associated shifts in cellular proportions, we identified novel cell type-specific biological insights into brain disorder associated regulatory variation. In most cases, genetic risk and epigenetic dysregulation targeted different cell types, thus suggesting independent mechanisms. For instance, Alzheimers disease genetic risk was exclusively enriched within microglia, while epigenetic dysregulation predominantly fell within oligodendrocyte-specific H3K27ac regions. In addition, reanalysis of the original datasets using CHAS enabled identification of biological pathways associated with each neurological and psychiatric disorder at cellular resolution.

genomics↗

bioRxiv2020

DNA methylation covariation in human whole blood and sperm: implications for studies of intergenerational epigenetic effects

BackgroundEpidemiological studies suggest that paternal obesity may increase the risk of fathering small for gestational age offspring. Studies in non-human mammals suggest that such associations could be mediated by DNA methylation changes in spermatozoa that influence offspring development in utero. Human obesity is associated with differential DNA methylation in peripheral blood. It is unclear, however, whether this differential DNA methylation is reflected in spermatozoa. We profiled genome-wide DNA methylation using the Illumina MethylationEPIC array in matched human blood and sperm from lean (discovery n=47; replication n=21) and obese (n=22) males to analyse tissue covariation of DNA methylation, and identify whether this covariation is influenced by obesity. ResultsDNA methylation signatures of human blood and spermatozoa are highly discordant, and methylation levels are correlated at only a minority of CpG sites ([~]1%). While at the majority of these sites, DNA methylation appears to be influenced by genetic variation, obesity-associated DNA methylation in blood was not generally reflected in spermatozoa, and obesity did not influence covariation patterns. However, one cross-tissue obesity-specific hypermethylated site (cg19357369; chr4:2429884; P=8.95 x 10-8; beta=0.02) was identified, warranting replication and further investigation. When compared to a wide range of human somatic tissue samples (n=5,917), spermatozoa displayed differential DNA methylation in pathways enriched in transcriptional regulation. ConclusionsHuman sperm displays a unique DNA methylation profile that is highly discordant to, and practically uncorrelated with, that of matched peripheral blood. Obesity only nominally influences sperm DNA methylation, making it an unlikely mediator of intergenerational effects of metabolic traits.

genomics↗