Biology subjects

Ichino, L.

Publications and source records attributed to Ichino, L..

3 recordsLinked to original sources

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↗