Biology subjects

Noviello, G.

Publications and source records attributed to Noviello, G..

7 recordsLinked to original sources

bioRxiv2026

Transcription-dependent heterochromatin at the Xist promoter shapes the random choice of the inactive X chromosome

In female mammals, Xist, the master regulator of X-chromosome inactivation (XCI), is expressed monoallelically. This pattern is established during early embryonic development, when the active Xist allele is chosen at random in each cell. How this choice is made remains incompletely understood. Combining knock-down and overexpression strategies in differentiating mouse embryonic stem cells, which recapitulate the onset of random XCI, we identify a role for the repressive chromatin mark H3K9me3 in the XCI initiation. We show that H3K9me3 accumulates at the promoter-proximal region of the silent Xist allele in female cells as monoallelic expression is established. Unexpectedly, this accumulation requires prior transcription of Xist itself, likely during the initial phase of upregulation, when Xist is frequently transcribed in male cells and from both X chromosomes in females. A repressive function of Xist-dependent H3K9me3 accumulation is supported by our finding that premature, transient Xist overexpression primes an allele for future silencing and skews the choice of the inactive X. Xist-dependent H3K9me3 recruitment does not require its antisense transcript Tsix, which can nonetheless enhance subsequent maintenance of the mark. In addition, the X-linked Xist activator RNF12 counteracts H3K9me3 formation, independently of its known target REX1. Our results thus point to facultative heterochromatin formation as a key contributor to choice at the onset of XCI, where activating and repressing mechanisms are intertwined to establish monoallelic Xist expression.

genetics↗

bioRxiv2025

HDAC3 prevents enhancer hyperactivation to enable developmental transitions

Dynamic gene regulation requires precise cooperation between transcription factors and chromatin modifiers at regulatory elements to achieve not only activation or repression, but also appropriate transcript dosage. However, the molecular mechanisms that ensure developmental genes are transcribed at specific levels remain largely unknown. Here, we discover that the epigenetic repressor histone deacetylase 3 (HDAC3), together with co-activators, binds a subset of the most active putative enhancer elements. Using a tunable mouse embryonic stem cell degron system, we uncover that HDAC3 directly prevents enhancer--and consequently gene--overactivation, effectively establishing a molecular "speed limit" for many naive pluripotency and housekeeping genes. Interestingly, we find that both the catalytic and non-catalytic functions of HDAC3 contribute to establishing this physiological transcript dose. Specifically during early development, HDAC3 rather than functioning as a canonical repressor, constrains the activity of highly transcribed genes of the implanting epiblast and ensures timely exit from naive pluripotency in vitro. Altogether, this indicates that a dynamic equilibrium between activators and repressors coexists at highly active enhancer elements in pluripotent stem cells, establishing appropriate transcriptional dosage and rendering them responsive to signaling cues, thereby enabling timely and coordinated developmental progression.

developmental biology↗

bioRxiv2024

Reporter CRISPR screens decipher cis- and trans-regulatory principles at the Xist locus

Developmental genes are controlled by an ensemble of cis-acting regulatory elements (REs), which in turn respond to multiple trans-acting transcription factors (TFs). Understanding how a cis-regulatory landscape integrates information from many dynamically expressed TFs has remained a challenge. We develop a combined CRISPR-screening approach using endogenous RNA and RE-reporters as readouts. Applied to the Xist locus, crucial for X-chromosome inactivation in females, this method allows us to comprehensively identify Xist-controlling TFs and map their TF-RE wiring. We find a group of transiently expressed TFs that regulate proximal REs, driving the binary activation of Xist expression. These basal activators are more highly expressed in cells with two X chromosomes, potentially driving female-specific Xist upregulation. A second set of developmental TFs is upregulated later during differentiation and targets distal REs. This regulatory axis is crucial to achieve high levels of Xist RNA, which is necessary for X-chromosome inactivation. Our findings support a model for developmental gene regulation in which factors targeting proximal REs drive binary ON-OFF decisions, while factors interacting with distal REs control the transcription output.

genetics↗

bioRxiv2024

Antisense transcription can induce expression memory via stable promoter repression

The capacity of cells to retain a memory of previous signals enables them to adopt unique cell fates and adjust to their surrounding environment. The underlying gene expression memory can arise from mutual repression of two genes, forming a toggle switch. Such mutual repression may occur at antisense loci, where two convergently oriented genes repress each other in cis. Under which conditions antisense transcription can generate expression memory remains poorly understood. To address this question, we combine mathematical modeling, genomics and a synthetic biology approach. Through simulations we show that stable memory can emerge, if both genes in an antisense pair transcribe through the convergent promoter and induce a stable repressive chromatin state. Genome-wide analysis of nascent transcription further supports antisense-mediated promoter repression with promoter-overlapping antisense gene pairs exhibiting mutually exclusive expression. Through constructing a synthetic antisense locus in mouse embryonic stem cells (mESCs) we then show that such a locus architecture can indeed maintain a memory of a transient stimulus. Mutual repression and the capacity for memory formation are elevated, when mESCs differentiate, showing that epigenetic memory is a cell type-specific property. Our finding that stem cells adapt their ability to remember stimuli as they differentiate might help to elucidate how stemness is maintained.

genetics↗

bioRxiv2022

CasTuner: a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression

Certain cellular processes are dose-dependent, requiring a specific quantity of gene products or a defined stoichiometry between them. This is exemplified by haploinsufficiency or by the need for dosage compensation for X-linked genes between the sexes in many species. Understanding dosage-sensitive processes requires the ability to perturb endogenous gene products in a quantitative manner. Here we present CasTuner, a CRISPR-based toolkit that allows analog tuning of endogenous gene expression. In the CasTuner system, activity of Cas-derived repressors is controlled through a FKBP12F36V degron domain and can thereby be quantitatively tuned by titrating the small molecule degrader dTAG-13. The toolkit can be applied at the transcriptional level, using the histone deacetylase hHDAC4 fused to dCas9, or at the post-transcriptional level, using the RNA-targeting CasRx. To optimise efficiency, inducibility and homogeneity of repression we target a fluorescently tagged endogenous gene, Esrrb, in mouse embryonic stem cells. Through flow cytometry, we show that CasTuner allows analog tuning of the target gene in a homogeneous manner across cells, as opposed to the widely used KRAB repressor domain, which exhibits a digital mode of action. We quantify repression and derepression dynamics for CasTuner and use it to measure dose-response curves between the pluripotency factor NANOG and several of its target genes, providing evidence for target-specific dose dependencies. CasTuner thus provides an easy-to-implement tool to perturb gene expression in an inducible, tunable and reversible manner and will be useful to study dose-responsive processes within their physiological context.

genetics↗

bioRxiv2022

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of a distal enhancer element

To ensure dosage compensation for X-linked genes between the sexes, one X chromosome is silenced during early embryonic development of female mammals. This process of X-chromosome inactivation (XCI) is initiated through upregulation of the RNA Xist from one X chromosome shortly after fertilization. Xist then mediates chromosome-wide gene silencing in cis and remains expressed in all cell types except the germ line and the pluripotent state, where XCI is reversed. The factors that drive Xist upregulation and thereby initiate XCI remain however unknown. We identify GATA transcription factors as potent Xist activators and demonstrate that they are essential for the activation of Xist in mice following fertilization. Through a pooled CRISPR activation screen we find that GATA1 can drive ectopic Xist expression in murine embryonic stem cells (mESCs). We demonstrate that all GATA factors can activate Xist directly via a GATA-responsive regulatory element (RE79) positioned 100 kb upstream of the Xist promoter. Additionally, GATA factors are essential for the induction of XCI in mouse preimplantation embryos, as simultaneous deletion of three members of the GATA family (GATA1/4/6) in mouse zygotes effectively prevents Xist upregulation. Thus, initiation of XCI and possibly its maintenance in distinct lineages of the preimplantation embryo is ensured by the combined activity of different GATA family members, and the absence of GATA factors in the pluripotent state likely contributes to X reactivation. We thus describe a form of regulation in which the combined action of numerous tissue-specific factors can achieve near-ubiquitous expression of a target gene.

genetics↗

bioRxiv2020

Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). In addition, TGS1 depletion results in increased hTR levels and telomere elongation in human cells. Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to devise new therapies for SMA and diseases caused by telomerase insufficiency.

genetics↗