Biology subjects

Mazzoni, E. O.

Publications and source records attributed to Mazzoni, E. O..

11 recordsLinked to original sources

bioRxiv2025

CTCF-RNA interactions orchestrate cell-specific chromatin loop organization

CCCTC-binding factor (CTCF) is essential for chromatin organization. CTCF interacts with endogenous RNAs, and deletion of its ZF1 RNA-binding region ({Delta}ZF1) disrupts chromatin loops in mouse embryonic stem cells (ESCs). However, the functional significance of CTCF-ZF1 RNA interactions during cell differentiation is unknown. Using an ESC-to-neural progenitor cell (NPC) differentiation model, we show that CTCF-ZF1 is crucial for maintaining cell-type-specific chromatin loops. Expression of CTCF-{Delta}ZF1 leads to disrupted loops and dysregulation of genes within these loops, particularly those involved in neuronal development and function. We identified NPC-specific, CTCF-ZF1 interacting RNAs. Truncation of two such coding RNAs, Podxl and Grb10, disrupted chromatin loops in cis, similar to the disruption seen in CTCF-{Delta}ZF1 expressing NPCs. These findings underscore the inherent importance of CTCF-ZF1 RNA interactions in preserving cell-specific genome structure and cellular identity. HIGHLIGHTSO_LICTCF loop anchors induced after differentiation are disrupted in the {Delta}ZF1 RNA-binding mutant. C_LIO_LILoop loss in the {Delta}ZF1 mutant is independent of its DNA binding and protein interactions. C_LIO_LIChromatin loop loss is associated with gene dysregulation. C_LIO_LITruncation of cell-specific, CTCF-ZF1-interacting RNAs disrupts chromatin loops in cis. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/643339v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1dad736org.highwire.dtl.DTLVardef@674a5dorg.highwire.dtl.DTLVardef@14345d4org.highwire.dtl.DTLVardef@19d83cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Molecular signatures of proteostasis control in motor neurons with different sensitivity to Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult neurodegenerative disease characterized by proteostasis dysregulation, resulting in progressive loss of spinal and upper motor neurons. A subset of cranial motor neurons resistant to ALS-stress survive until late stages of the disease. To investigate these differences, we exploited a unique platform of induced cranial and spinal motor neurons (iCrMNs and iSpMNs, respectively). Exposing both cell types to proteotoxic stress, we quantified transcriptome and proteome changes over 36 hours for a core set of >8,200 genes. While mRNA and protein changes under stress were congruent for many genes, cell-type specific differences manifested at either the RNA or protein level, but less at both. At the protein level, iCrMNs and iSpMNs differed significantly with respect to abundance of many membrane proteins, including synaptic proteins, solute carriers, adhesion molecules, and signaling molecules suggesting that the superior stress survival of iCrMNs involve diverse pathways supporting neuronal function. Other differences included genes involved in ribosome biogenesis and subunits of the core proteasome. We investigated the role of proteasomal degradation in more detail. Our data showed that although stress reduces proteasome activity in both neuronal types, iCrMNs had significantly more abundant and active 26S proteasome than iSpMNs, which indicate a higher capacity for the degradation of ubiquitinated proteins. We identified a new regulator of this better performance, i.e. the nuclear proteasome activator Ublcp1, whose inhibition sensitized iCrMNs, but not iSpMNs, to stress and abolished their higher survival rates. The results suggest that the two neuronal cell types regulate and use the degradation machinery differently under normal and stress conditions. Overall, this work demonstrates the value of unbiased system-wide analyses in generating hypotheses on differential proteostasis regulation in cranial and spinal motor neurons.

systems biology↗

bioRxiv2022

ALS sensitive spinal motor neurons enter a degenerative downward spiral of impaired splicing and proteostasis

Despite clear therapeutic potential, the mechanisms that confer differential neuronal sensitivity are not well understood. During Amyotrophic Lateral Sclerosis (ALS), sensitive spinal motor neurons (SpMN) die while a subset of rostral cranial motor neurons (CrMN) survive. In this work, we optimized a protocol to differentiate CrMNs and SpMNs from human induced pluripotent stem cells (iPSCs) by direct programming and positional patterning. Human iCrMNs are more resistant than iSpMNs to proteotoxic stress and rely on the proteasome to maintain proteostasis. iCrMNs better prevent mislocalization of TDP43 from the nucleus, a hallmark of ALS progression. iSpMNs contain more splicing defects than iCrMNs in response to ALS-related stress with genes involved in splicing and proteostasis maintenance. Therefore, iCrMNs resist ALS at two levels, preventing protein accumulation and reducing splicing defects in response to TDP43 nuclear depletion. Thus, ALS-sensitive iSpMNs appear to enter a downward spiral compromising their ability to maintain proteostasis and splicing.

neuroscience↗

bioRxiv2021

PRC1 Sustains the Memory of Neuronal Fate Independent of PRC2 Function

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We unexpectedly found that PRC2 is dispensable to preserve the morphogen-induced positional fates of spinal motor neurons (MNs), while PRC1 is essential for the specification of segmentally-restricted subtypes. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox networks by derepressed caudal Hox genes. These results indicate that PRC1 can function independently of de novo PRC2-dependent histone methylation to maintain chromatin topology and transcriptional memory at the time of neuronal differentiation.

neuroscience↗

bioRxiv2021

Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modes controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce synthetic regulatory reconstitution, a novel conceptual framework for studying gene regulation and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130-170 kilobases each) to an ectopic location in the mouse genome. We find that a HoxA cluster lacking distal enhancers recapitulates correct patterns of chromatin remodeling and transcription in response to patterning signals, while distal enhancers are required for full transcriptional output. Synthetic regulatory reconstitution is a generalizable strategy to decipher the regulatory logic of gene expression in complex genomes. One-Sentence SummaryReconstitution of gene regulation using large DNA constructs unravels the regulatory logic of a developmental gene locus.

synthetic biology↗

bioRxiv2020

The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by pausing POL II atpro-differentiation genes

In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ~1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ZBTB11 and ZFP131 maintain promoter-proximally paused Polymerase II at pro-differentiation genes in ESCs. ZBTB11 or ZFP131 loss leads to NELF pausing factor release, an increase in H3K4me3, and transcriptional upregulation of genes associated with all three germ layers. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency. One-sentence summaryA Transcription Factor-wide CRISPR screen identifies ZBTB11 and ZFP131 maintaining pluripotency by pausing POL II at pro-differentiation genes

developmental biology↗

bioRxiv2020

A CRISPR Screen Identifies Myc-associated Zinc Finger Protein (MAZ) as an Insulator Functioning at CTCF boundaries in Hox Clusters

The essential CCCTC-binding factor (CTCF) is critical to three-dimensional (3D) genome organization. CTCF binding insulates active and repressed genes within the Hox clusters upon differentiation, but such binding does not participate in boundary formation in all cell types, such as embryonic stem cells. We conducted a genome-wide CRISPR knockout screen to identify genes required for CTCF boundary activity at the HoxA cluster, complemented by novel biochemical approaches. This screen identified Myc-associated zinc finger protein (MAZ) as a CTCF insulator co-factor, among other candidates listed herein. MAZ depletion or specific deletion of MAZ motifs within the Hox clusters led to de-repression of posterior Hox genes immediately after CTCF boundaries upon differentiation, which phenocopied deletion of the proximal CTCF motifs. Similar to CTCF, MAZ interacted with the cohesin subunit, RAD21. Upon loss of MAZ, local contacts within topologically associated domains (TADs) were disrupted, as evidenced by HiC analysis. Thus, MAZ is a novel factor sharing insulation properties with CTCF and contributing to the genomic architectural organization. One Sentence SummaryMAZ is identified as an insulator functioning at CTCF boundaries delimiting active and repressed genes at Hox clusters

biochemistry↗

bioRxiv2019

Hox binding specificity is directed by DNA sequence preferences and differential abilities to engage inaccessible chromatin.

While Hox genes encode for conserved transcription factors (TFs), they are further divided into anterior, central, and posterior groups based on their DNA-binding domain similarity. The posterior Hox group expanded in the deuterostome clade and patterns caudal and distal structures. We aim to address how similar HOX TFs diverge to induce different positional identities. We studied HOX TF DNA-binding and regulatory activity during an in vitro motor neuron differentiation system that recapitulates embryonic development. We find diversity in the genomic binding profiles of different HOX TFs, even among the posterior group paralogs that share similar DNA binding domains. These differences in genomic binding are explained by differing abilities to bind to previously inaccessible sites. For example, the posterior group HOXC9 has a greater ability to bind occluded sites than the posterior HOXC10, producing different binding patterns and driving differential gene expression programs. From these results, we propose that the differential abilities of posterior HOX TFs to bind to previously inaccessible chromatin drive patterning diversification.

developmental biology↗

bioRxiv2019

A simple method to quantify proteins from one thousand cells

The rise of single-cell transcriptomics has created an urgent need for similar approaches that use a minimal number of cells to quantify expression levels of proteins. We integrated and optimized multiple recent developments to establish a proteomics workflow to quantify proteins from as few as 1,000 mammalian stem cells. The method uses chemical peptide labeling, does not require specific equipment other than cell lysis tools, and quantifies >2,500 proteins with high reproducibility. We validated the method by comparing mouse embryonic stem cells and in vitro differentiated motor neurons. We identify differentially expressed proteins with small fold-changes, and a dynamic range in abundance similar to that of standard methods. Protein abundance measurements obtained with our protocol compare well to corresponding transcript abundance and to measurements using standard inputs. The protocol is also applicable to other systems, such as FACS-purified cells from the tunicate Ciona. Therefore, we offer a straightforward and accurate method to acquire proteomics data from minimal input samples.

systems biology↗

bioRxiv2019

Characterizing the sequence and prior chromatin determinants of induced TF binding with bimodal neural networks

Transcription factor (TF) binding specificity is determined via a complex interplay between the TFs DNA binding preference and cell type-specific chromatin environments. The chromatin features that correlate with TF binding in a given cell type have been well characterized. For instance, the binding sites for a majority of TFs display concurrent chromatin accessibility. However, concurrent chromatin features reflect the binding activities of the TF itself, and thus provide limited insight into how genome-wide TF-DNA binding patterns became established in the first place. To understand the determinants of TF binding specificity, we therefore need to examine how newly activated TFs interact with sequence and preexisting chromatin landscapes. Here, we investigate the sequence and preexisting chromatin predictors of TF-DNA binding by examining the genome-wide occupancy of TFs that have been induced in well-characterized chromatin environments. We develop Bichrom, a bimodal neural network that jointly models sequence and preexisting chromatin data to interpret the genome-wide binding patterns of induced TFs. We find that the preexisting chromatin landscape is a differential global predictor of TF-DNA binding; incorporating preexisting chromatin features improves our ability to explain the binding specificity of some TFs substantially, but not others. Furthermore, by analyzing site-level predictors, we show that TF binding in previously inaccessible chromatin tends to correspond to the presence of more favorable cognate DNA sequences. Bichrom thus provides a framework for modeling, interpreting, and visualizing the joint sequence and chromatin landscapes that determine TF-DNA binding dynamics.

bioinformatics↗

bioRxiv2017

Characterizing regulatory sequence features that discriminate between overlapping annotation labels

Genomic loci with regulatory potential can be identified and annotated with various properties. For example, genomic sites may be annotated as being bound by a given transcription factor (TF) in one or more cell types. The same sites may be further labeled as being proximal or distal to known promoters. Given such a collection of labeled sites, it is natural to ask what sequence features are associated with each annotation label. However, discovering such label-specific sequence features is often confounded by overlaps between annotation labels; e.g. if regulatory sites specific to a given cell type are also more likely to be promoter-proximal, it is difficult to assess whether motifs identified in that set of sites are associated with the cell type or associated with promoters. In order to meet this challenge, we developed SeqUnwinder, a principled approach to deconvolving interpretable discriminative sequence features associated with overlapping annotation labels. We demonstrate the novel analysis abilities of SeqUnwinder using three examples. Firstly, we show SeqUnwinders ability to unravel sequence features associated with the dynamic binding behavior of TFs during motor neuron programming from features associated with chromatin state in the initial embryonic stem cells. Secondly, we characterize distinct sequence properties of multi-condition and cell-specific TF binding sites after controlling for uneven associations with promoter proximity. Finally, we demonstrate the scalability of SeqUnwinder to discover cell-specific sequence features from over one hundred thousand genomic loci that display DNase I hypersensitivity in one or more ENCODE cell lines.\n\nAvailabilityhttps://github.com/seqcode/sequnwinder

bioinformatics↗