Biology subjects

Fazzio, T. G.

Publications and source records attributed to Fazzio, T. G..

11 recordsLinked to original sources

bioRxiv2025

esBAF and INO80C fine-tune subcompartments and differentially regulate enhancer-promoter interactions

The genome is compacted in the nucleus through a hierarchical chromatin organization, ranging from chromosome territories to compartments, topologically associating domains (TADs), and individual nucleosomes. Nucleosome remodeling complexes hydrolyze ATP to translocate DNA and thereby mobilize histone proteins. While nucleosome remodeling complexes have been extensively studied for their roles in regulating nucleosome positioning and accessibility, their contributions to higher-order chromatin architecture remain less well understood. Here, we investigate the roles of two key nucleosome remodelers, esBAF and INO80C, in shaping 3D genome organization in mouse embryonic stem cells. Using Hi-C, we find that loss of either remodeler has minimal effects on global compartment or TAD structures. In contrast, subcompartment organization is notably altered, suggesting that esBAF and INO80C contribute to finer-scale chromatin topology. To overcome the limited resolution of Hi-C for detecting regulatory loops, we employed promoter capture Micro-C (PCMC), which revealed that the loss of esBAF or INO80C alters a subset of promoter anchored looping interactions. Although these changes occur at distinct genomic loci for each remodeler, the affected sites are commonly enriched for bivalent chromatin regions bound by OCT4, SOX2, and NANOG (OSN), as well as BRG1 and INO80 themselves. Together, our findings reveal that esBAF and INO80C selectively influence subcompartment identity and enhancer-promoter communication at key regulatory loci, highlighting a previously underappreciated role for nucleosome remodelers in higher-order chromatin organization.

genomics↗

bioRxiv2025

R-loops shape chromatin architecture to promote balanced lineage allocation during differentiation

R-loops--RNA:DNA hybrids that often form co-transcriptionally--are emerging as key regulators of genome function, yet their roles in shaping chromatin architecture and developmental potential remain incompletely defined. Here, we use inducible Rnaseh1 expression in mouse embryonic stem cells (mESCs) to achieve acute, global R-loop depletion and systematically interrogate their impact on chromatin structure and lineage specification. We find that R-loop loss has minimal effect on steady-state gene expression or self-renewal. Instead, it leads to a striking reduction in H2A.Z occupancy at both active and bivalent promoters, accompanied by increased nucleosome density--revealing a previously unrecognized role for R-loops in maintaining promoter architecture. During gastruloid differentiation, R-loop-depleted mESCs exhibit a pronounced bias toward ectodermal fates, along with dysregulation of lineage-specific transcription factors and impaired cell-cell signaling. Consistent with these alterations, R-loop-depleted cells show widespread perturbations in gene regulatory networks across several early cell types. These findings uncover a critical role for R-loops in shaping the H2A.Z chromatin landscape and preserving balanced lineage trajectories during early development, offering new insights into the epigenomic regulation of stem cell fate.

genomics↗

bioRxiv2024

PRMT5 orchestrates EGFR and AKT networks to activate NFκB and promote EMT

Neuroblastoma remains a formidable challenge in pediatric oncology, representing 15% of cancer-related mortalities in children. Despite advancements in combinatorial and targeted treatments improving survival rates, nearly 50% of patients with high-risk neuroblastoma will ultimately succumb to their disease. Dysregulation of the epithelial-mesenchymal transition (EMT) is a key mechanism of tumor cell dissemination, resulting in metastasis and poor outcomes in many cancers. Our prior work identified PRMT5 as a key regulator of EMT via methylation of AKT at arginine 15, enhancing the expression of EMT-driving transcription factors and facilitating metastasis. Here, we identify that PRMT5 directly regulates the transcription of the epidermal growth factor receptor (EGFR). PRMT5, through independent modulation of the EGFR and AKT pathways, orchestrates the activation of NF{kappa}B, resulting in the upregulation of the pro-EMT transcription factors ZEB1, SNAIL, and TWIST1. Notably, EGFR and AKT form a compensatory feedback loop, reinforcing the expression of these EMT transcription factors. Small molecule inhibition of PRMT5 methyltransferase activity disrupts EGFR/AKT signaling, suppresses EMT transcription factor expression and ablates tumor growth in vivo. Our findings underscore the pivotal role of PRMT5 in the control of the EMT program in high-risk neuroblastoma.

cancer biology↗

bioRxiv2022

Revisiting chromatin packaging in mouse sperm

Mammalian sperm exhibit an unusual and heavily-compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, histone localization, histone modification, and chromosome folding. Here, we revisit these studies in light of recent reports that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis we readily recapitulate multiple prominent genome-wide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, along with appropriate lysis conditions, are required to reveal a sperm chromatin state distinct from most previous reports. Using ATAC-Seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with early development and transcriptional control. Histone modification and chromosome folding studies also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies strongly argue that our knowledge of mammalian chromosome packaging remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm.

molecular biology↗

bioRxiv2022

Phosphosite Scanning reveals a complex phosphorylation code underlying CDK-dependent activation of Hcm1

Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates processive phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.

cell biology↗

bioRxiv2022

EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance

T cell-activating immunotherapies that produce durable and even curative responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of cellular senescence and its accompanying senescence-associated secretory phenotype (SASP) can be an effective approach to activate not only T cell but also cytotoxic Natural Killer (NK) cell-mediated anti-tumor immunity. Here we found that the pancreas tumor microenvironment (TME) suppresses NK and T cell surveillance following therapy-induced senescence through EZH2-mediated repression of pro-inflammatory SASP genes. Genetic or pharmacological inhibition of EZH2 or its methyltransferase activity stimulated the production of pro-inflammatory SASP chemokines CCL2 and CXCL9/10 that led to enhanced NK and T cell infiltration and tumor eradication in preclinical PDAC mouse models. EZH2 activity was also associated with suppression of SASP-associated inflammatory chemokines and cytotoxic lymphocyte immunity and reduced overall survival in a PDAC patient cohort. These results demonstrate that EZH2 mediates epigenetic repression of the pro-inflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to potentiate NK and T cell surveillance in PDAC to achieve immune-mediated tumor control.

cancer biology↗

bioRxiv2022

S-adenosylmethionine synthases specify distinct H3K4me3 populations and gene expression patterns during heat stress

Methylation is a widely occurring modification that requires the methyl donor S-adenosylmethionine (SAM) and acts in regulation of gene expression and other processes. SAM is synthesized from methionine, which is imported or generated through the 1-carbon cycle (1CC). Alterations in 1CC function have clear effects on lifespan and stress responses, but the wide distribution of this modification has made identification of specific mechanistic links difficult. Exploiting a dynamic stress-induced transcription model, we find that two SAM synthases in Caenorhabditis elegans, SAMS-1 and SAMS-4, contribute differently to modification of H3K4me3, gene expression and survival. We find that sams-4 enhances H3K4me3 in heat shocked animals lacking sams-1, however, sams-1 cannot compensate for sams-4, which is required to survive heat stress. This suggests that the regulatory functions of SAM depend on its enzymatic source and that provisioning of SAM may be an important regulatory step linking 1CC function to phenotypes in aging and stress.

cell biology↗

bioRxiv2021

TAF4b transcription networks regulating early oocyte differentiation

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here we use a combination of gene expression profiling and chromatin mapping to characterize the TAF4b gene regulatory network in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin, and X-linked genes, and unexpectedly we found a connection with Turner Syndrome pathways. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in meiosis and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/KLF family motifs rather than TATA-box, suggesting an alternate mode of promoter interaction. Together, our data connects several gene regulatory nodes that contribute to the ovarian reserve.

developmental biology↗

bioRxiv2021

Simultaneous profiling of multiple chromatin proteins in the same cells

Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of colocalization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and characterization of cell type-specific chromatin architecture. In sum, multi-CUT&Tag increases the information content per cell of epigenomic maps, facilitating direct analysis of the interplay of different proteins on chromatin.

genomics↗

bioRxiv2019

MTF1, a classic metal sensing transcription factor, promotes myogenesis in response to copper.

MTF1 is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements (MREs). MTF1 responds to metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. We used multiple strategies to identify an unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, MTF1 expression increased, as did nuclear localization. Mtf1 knockdown impaired differentiation, while addition of non-toxic concentrations of Cu+ enhanced MTF1 expression and promoted myogenesis. Cu+ bound stoichiometrically to a C-terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes and binding was stimulated by copper. MTF1 formed a complex with MyoD at myogenic promoters, the master transcriptional regulator of the myogenic lineage. These studies establish novel mechanisms by which copper and MTF1 regulate gene expression in myoblast differentiation.

cell biology↗

bioRxiv2018

Profiling of pluripotency factors in individual stem cells and early embryos

Major cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small cell populations within developing embryos. To understand how TFs regulate cell fate it is important to identify their genomic binding sites in these populations. However, current methods cannot profile TFs genome-wide at or near the single cell level. Here we adapt the CUT&RUN method to profile chromatin proteins in low cell numbers, mapping TF-DNA interactions in single cells and individual pre-implantation embryos for the first time. Using this method, we demonstrate that the pluripotency TF NANOG is significantly more dependent on the SWI/SNF family ATPase BRG1 for association with its genomic targets in vivo than in cultured cells--a finding that could not have been made using traditional approaches. Ultra-low input CUT&RUN (uliCUT&RUN) enables interrogation of TF binding from low cell numbers, with broad applicability to rare cell populations of importance in development or disease.

genomics↗