Biology subjects

Ichino, L.

Publications and source records attributed to Ichino, L..

7 recordsLinked to original sources

bioRxiv2026

Enhancer Tuning by Sequence-Specific Repressors

Enhancers integrate combinatorial inputs from sequence-specific transcription factors (TFs) and their activity must be calibrated to achieve precise spatiotemporal control of transcript dosage. Here we demonstrate that the sequence-specific repressors SNAI1 and SNAI2 (i.e. SNAIL and SLUG) quantitatively tune enhancer activity. In human neural crest cells, SNAI1/2 occupy a subset of active enhancers, where their depletion increases H3K27ac, chromatin accessibility, and enhancer regulatory potential. Changes in SNAI1/2 binding motifs contribute to enhancer divergence between human and chimpanzee, suggesting an evolutionary role for the repressor-mediated tuning. Single-molecule chromatin profiling using Deaminase-Assisted Fiber-seq (DAF-seq) reveals that individual enhancers toggle between an ensemble of open and nucleosome-dense chromatin states. While transcriptional activators increase the fraction of the open states, SNAI1/2 shift the equilibrium toward nucleosome-occupied states. This impedes binding of activator TFs, without fully repressing the enhancer. We propose that SNAI1/2 function as a molecular dimmer switch, modulating nucleosome dynamics to calibrate enhancer output.

molecular biology↗

bioRxiv2026

Enhancer buffering protects dosage-sensitive housekeeping genes during vulnerable developmental transitions

Housekeeping genes maintain robust expression across cell types despite dynamic transcription factor fluctuations, yet their haploinsufficiency is associated with many tissue-specific developmental disorders. To understand this paradox, we focus on TCOF1, a broadly expressed regulator of rRNA synthesis, whose haploinsufficiency causes Treacher Collins syndrome (TCS). We show that transitional cranial neural crest cells (tCNCC) undergoing mesenchymal specification exhibit extreme dosage-sensitivity to TCOF1, but not to RNA polymerase I, explaining both cellular origins of TCS and prevalence of TCOF1 mutations in the disease. To maintain robust expression, TCOF1 deploys CNCC-specific enhancers that buffer against fluctuations in promoter-regulating factors, converting sensitive expression responses into threshold-protected outputs. Systematic promoter-enhancer coupling experiments demonstrate this principle generalizes across many dosage-sensitive housekeeping genes, whose promoters operate near saturation and only reveal their enhancer-dependency under suboptimal conditions. Thus, enhancers play a unique role at housekeeping promoters: rather than amplifying expression, they ensure its robustness during vulnerable developmental transitions.

genetics↗

bioRxiv2025

An antagonistic epigenetic mechanism regulating gene expression in pollen revealed through single-nucleus multiomics

Arabidopsis MBD5, MBD6, and MBD7 are CG-specific methyl-readers with opposite functions: MBD5 and MBD6 (MBD5/6) repress methylated loci in pollen vegetative nuclei (VN), while MBD7 prevents transgene silencing, possibly by promoting DNA demethylation. Here we show that loss of MBD7 rescues transcriptional defects at a large subset of MBD5/6-bound loci. Using simultaneous profiling of DNA methylation and transcription in single pollen nuclei, we found that MBD5/6-bound loci that are actively demethylated in immature VN lose additional methylation in mbd5/6, prior to transcriptional derepression. A subset of these loci is also bound by MBD7, correlating with demethylation and transcriptional derepression in mbd5/6 that are both reversed by loss of MBD7. Conversely, ectopically recruiting the MBD7 complex to MBD5/6 targets causes partial demethylation and upregulation. We propose that MBD5/6 maintain silencing in VN in part by preventing the MBD7 complex from enhancing the active demethylation that occurs during VN maturation.

molecular biology↗

bioRxiv2023

MBD2 couples DNA methylation to Transposable Elements silencing during male gametogenesis.

DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2, and MBD4, and show that MBD2 acts as a transposable element (TE) repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused TE activation in the vegetative cell (VC) of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. Loss of silencing in mbd2 became more significant in the mbd5 mbd6 or adcp1 mutant backgrounds, as well as in plants with chemically induced genome-wide DNA demethylation, suggesting that MBD2 acts redundantly with other silencing pathways to safeguard TEs from activation. Overall, our study identifies MBD2 as a novel methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.

plant biology↗

bioRxiv2023

ACD15, ACD21, and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements.

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG-binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here we characterize two additional complex members: -crystalline domain containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of complex components regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of gene silencing complexes that act to drive the formation of higher order, dynamic assemblies. One-Sentence SummaryArabidopsis ACD21 and ACD15 drive accumulation of MBD5/6 complex silencing assemblies at methyl-CG sites and recruit SLN to maintain protein mobility in these assemblages.

cell biology↗

bioRxiv2023

H1 restricts euchromatin-associated methylation pathways from heterochromatic encroachment

Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in h1, suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.

plant biology↗

bioRxiv2022

Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing during epigenetic reprogramming in pollen

Silencing of transposable elements (TEs) drove the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here we show via single-nucleus RNA-seq of developing male gametophytes that these repressors are critical for TE silencing in the pollen vegetative cell, which undergoes epigenetic reprogramming causing chromatin decompaction to support fertilization by sperm cells. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We found that TEs repressed by MBD5/6 gain accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system especially important when chromatin compaction is compromised.

molecular biology↗