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A lamina-associated domain border governs nuclear lamina interactions, transcription and recombination of the Tcrb locus [ChIP-seq]

ABSTRACT: Tcrb locus V(D)J recombination is regulated by positioning at the nuclear periphery. Here we used DamID to profile Tcrb locus interactions with the nuclear lamina at high-resolution. We identified a lamina-associated domain (LAD) border composed of several CTCF binding elements that segregates active non-LAD from inactive LAD regions of the locus. Deletion of the LAD border caused an enhancer-dependent spread of H3K27ac from the active recombination center into recombination center-proximal LAD chromatin. This was associated with a disruption to LAD organization, increased chromatin looping to the recombination center, and increased transcription and recombination of recombination center-proximal gene segments. Our results show that a Tcrb locus LAD and LAD border are critical components of Tcrb locus gene regulation and suggest that LAD borders may generally function to constrain the activity of nearby enhancers.

ORGANISM(S): Mus musculus

PROVIDER: GSE115582 | GEO | 2018/10/22

REPOSITORIES: GEO

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A lamina-associated domain border governs nuclear lamina interactions, transcription and recombination of the Tcrb locus [DamID]

Project description:Tcrb locus V(D)J recombination is regulated by positioning at the nuclear periphery. Here we used DamID to profile Tcrb locus interactions with the nuclear lamina at high-resolution. We identified a lamina-associated domain (LAD) border composed of several CTCF binding elements that segregates active non-LAD from inactive LAD regions of the locus. Deletion of the LAD border caused an enhancer-dependent spread of H3K27ac from the active recombination center into recombination center-proximal LAD chromatin. This was associated with a disruption to LAD organization, increased chromatin looping to the recombination center, and increased transcription and recombination of recombination center-proximal gene segments. Our results show that a Tcrb locus LAD and LAD border are critical components of Tcrb locus gene regulation and suggest that LAD borders may generally function to constrain the activity of nearby enhancers.

2018-10-22 | GSE116953 | GEO

A lamina-associated domain border governs nuclear lamina interactions, transcription and recombination of the Tcrb locus.

Project description:A lamina-associated domain border governs nuclear lamina interactions, transcription and recombination of the Tcrb locus.

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A lamina-associated domain border governs nuclear lamina interactions, transcription and recombination of the Tcrb locus [DamID]

Project description:A lamina-associated domain border governs nuclear lamina interactions, transcription and recombination of the Tcrb locus [DamID]

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A lamina-associated domain border governs nuclear lamina interactions, transcription and recombination of the Tcrb locus [ChIP-seq]

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Mapping the micro-proteome of the nuclear lamina and lamin associated domains

Project description:The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, so-called lamin associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina as well as identified putative LAD proximal proteins. Our analysis identifies many heterochromatin related proteins related to H3K9 methylation processes as well as many proteins related to cell cycle regulation identifying important proteins essential for LAD function.

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Decoding the determinants of genome-nuclear lamina interactions by a random scrambling approach (RNA-Seq)

Project description:Mammalian genomes contain hundreds of lamina-associated domains (LADs), which are large, often megabase-sized heterochromatin domains that are anchored to the nuclear lamina (NL). Even though LADs play a key role in the spatial organization of the genome and potently repress gene activity, it is not yet understood how their interactions with the NL are encoded in their DNA. Here, we investigated the principles that govern LAD-NL interactions by taking a "LAD scrambling" approach. This approach relies on 1) local "hopping" of a Sleeping Beauty transposon to randomly insert loxP sites within and around a LAD of choice; and 2) Cre-mediated recombination between the loxP sites. This approach is highly efficient, enabling us to establish a large collection of clonal cell lines with deletions and inversions up to ~2Mb in size, spanning LAD and their flanking inter-LAD sequences. Mapping of NL interactions in these clones revealed that a single LAD contacts the NL through multiple regions. These regions act cooperatively and redundantly; however, some have more affinity for the NL and can boost NL contacts of neighbouring sequences. We also observed a crosstalk between two neighbouring LADs, when close enough to each other. Finally, changes in the heterochromatin mark H3K9me3 only partially mirrored changes in NL contacts. They also correlated more with gene expression changes than NL contacts did. These principles of LAD - NL interactions provide insight into the overall architecture of the genome and the impact on gene regulation.

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Decoding the determinants of genome-nuclear lamina interactions by a random scrambling approach (pA-DamID)

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Project description:Topologically Associating Domains (TADs) partition the genome into self-interacting regions through boundaries. Chromatin configuration plays a crucial role in TCR gene rearrangements, thereby affecting repertoire diversity. To assess the dynamics of chromatin boundaries within TCR genes, we conducted Hi-C assays on double-negative (DN) and double-positive (DP) cells from RAG-deficient mice. A chromatin boundary at the 3' end of the Tcrb locus observed in DN cells dissipated in DP cells, thereby altering interactions between the main Vβ cluster and the recombination center. Additionally, the INTs boundary in the Tcra-Tcrd locus disappeared in DP cells, leading to enhanced interactions between the proximal Vα region and recombination center at the Jα segments. Subsequent analysis revealed that the disappearance of boundaries was not attributable to diminished CTCF binding but rather heightened activity within these regions. This study illuminates the developmental dynamics of TAD boundaries in TCR genes, enriching our understanding of their evolving nature.

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Regulatory T cells from colonic lamina propria [array]

Project description:The colonic lamina propria contains a distinct population of Foxp3+ T regulatory cells (Tregs) that modulate responses to commensal microbes. Analysis of gene expression revealed that the transcriptome of colonic Tregs is distinct from splenic and other tissue Tregs. Rorγ and Helios in colonic Tregs mark distinct populations: Rorγ+Helios- or Rorγ-Helios+ Tregs. We uncovered an unanticipated role for Rorγ, a transcription factor generally considered to be antagonistic to Foxp3. Rorγ in colonic Tregs accounts for a small but specific part of the colon-specific Treg signature. (1) Total colonic and splenic Foxp3+ Treg comparison: Lymphocytes were isolated from colonic lamina propria and spleens of Foxp3-ires-GFP mice, where GFP reports Foxp3 expression. TCRb+CD4+GFP+ cells were double sorted into Trizol. (2) Colonic Rorγ+ and Rorγ- Treg comparison: Foxp3-ires-Thy1.1 reporter mice were crossed to Rorc-GFP reporter mice to generate mice that report both Foxp3 and Rorγ expression. Rorγ+Foxp3+ Tregs (TCRb+CD4+Thy1.1+GFP+) and Rorγ-Foxp3+ Tregs (TCRb+CD4+Thy1.1+GFP-) from colonic lamina propria were double sorted into Trizol.To reduce variability and increase cell number, cells from multiple mice were pooled for sorting and at least three replicates were generated for all groups. RNA from 1.5-3.0 x104 cells was amplified, labeled and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays.

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