Biology subjects

Korthout, T.

Publications and source records attributed to Korthout, T..

5 recordsLinked to original sources

bioRxiv2023

Time-resolved and multifactorial profiling in single cells resolves the order of heterochromatin formation events during X-chromosome inactivation

The regulation of gene expression is governed at multiple levels of chromatin organization. However, how coordination is achieved remains relatively unexplored. Here we present Dam&ChIC, a method that enables time-resolved and multifactorial chromatin profiling at high resolution in single cells. Analysis of genome-lamina interactions in haploid cells reveals highly dynamic spatial repositioning of small domains during interphase and partial inheritance over mitosis. Dam&ChIC applied to study random X-inactivation uncovers that spreading of H3K27me3 on the inactive X chromosome (Xi) overlaps with remarkable genome-lamina detachment. We find that genome-lamina detachment precedes H3K27me3 accumulation on the Xi and occurs upon mitotic exit. Domains that retain genome-lamina interactions are marked by high pre-existing H3K9me3 levels. These findings imply an important role for genome-lamina interactions in regulating H3K27me3 accumulation on the Xi. We anticipate that Dam&ChIC will be instrumental in unraveling the interconnectivity and order of chromatin events underlying cell-state changes in single cells.

genomics↗

bioRxiv2023

Combinatorial single-cell profiling of all major chromatin types with MAbID

Gene expression programs result from the collective activity of many regulatory factors. To obtain insight into the mechanisms that govern gene regulation, it is imperative to study their combined mode of action and interconnectivity. However, it has been challenging to simultaneously measure a combination of these factors within one sample. Here, we introduce MAbID, a method that combines genomic profiling of many histone modifications and chromatin-binding proteins in a single reaction. MAbID employs antibody-DNA conjugates to enable genomic barcoding of chromatin at sites of epitope occupancy. This barcoding strategy allows for the combined incubation of multiple antibodies in a single sample to reveal the genomic distributions of many epigenetic states simultaneously. We used MAbID to profile both active and inactive chromatin types in human cell lines and multiplexed measurements in the same sample without loss of data quality. Moreover, we obtained joint measurements of six epitopes covering all major chromatin types in single cells during mouse in vitro neural differentiation and captured associated changes in multifactorial chromatin states. Thus, MAbID holds the potential to gain unique insights into the interplay between gene regulatory mechanisms, especially in settings with limited sample material and in single cells.

genomics↗

bioRxiv2019

The histone methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+ T cells

Cytotoxic T-cell differentiation is guided by epigenome adaptations but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T-cell specific ablation of Dot1L resulted in loss of naive CD8+ T cells and premature differentiation towards a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Without DOT1L, the memory-like CD8+ cells fail to acquire full effector functions in vitro and in vivo. Mechanistically, DOT1L controlled T-cell differentiation and function by ensuring normal T-cell receptor density and signaling, and by maintaining epigenetic identity, in part by indirectly supporting the repression of developmentally-regulated genes. Through our study DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and supporting the licensing of the full effector potential of cytotoxic T cells.

immunology↗

bioRxiv2019

Evolutionarily-conserved chromatin crosstalk generates a DOT1L-dose dependency in thymic lymphoma caused by loss of HDAC1

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL-rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia but the cellular mechanisms that regulate DOT1L are still poorly understood. Here we identify the budding yeast histone deacetylase Rpd3 as a negative regulator of Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1{Delta}/{Delta} thymic lymphomas were sensitive to DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily-conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.

cell biology↗

bioRxiv2017

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Transcription, replication and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein-DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-ChIP-Barcode-Seq technology in budding yeast to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 proteins. Moreover, replication stress induced changes in local chromatin-proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Epi-Decoder will enable the delineation of complex and dynamic protein-DNA interactions across many regions of the genome.

genomics↗