Genome organization by SATB1 binding to base-unpairing regions (BURs) provides a scaffold for SATB1-regulated gene expression

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    eLife Assessment

    This important study has modified ChIP-seq and 4C-seq procedures with a urea step and shows that this drastically changes the pattern of chromatin interactions observed for SATB1 but not other proteins (CTCF, Jarid2, Suz12, Ezh2). Multiple controls make the data convincing. The findings shed new light on the role of SATB1 in genome organization and will be of interest to those who study chromosome structure and nuclear organization.

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Abstract

Mammalian genomes are organized by multi-level folding, yet how this organization contributes to cell type-specific transcription remain unclear. We uncovered that the nuclear protein SATB1 establishes two-tiered chromatin organization, one through indirect binding and another by direct binding of base-unpairing regions (BURs), which are genomic elements with high unwinding propensities. Published ChIP-seq datasets show SATB1 binding to highly accessible chromatin at enhancers and CTCF sites, but not to BURs. By employing urea ChIP-seq, which retains only directly bound protein:DNA complexes, we found that BURs, but not CTCF sites, are direct SATB1 binding targets. SATB1-bound BUR interactions with accessible chromatin can cross multiple topologically associated domains (TADs) and SATB1 is required for these megabase-scale interactions linked to cell type-specific gene expression. BURs are mainly found within lamina associated domains (LADs) sequestered at the nuclear lamina, but also in inter-LADs, and SATB1 binds a subset of BURs depending on cell type. Notably, despite the mutually exclusive SATB1-binding profiles uncovered by the two ChIP-seq methods, we found most peaks in both profiles are real and require SATB1. Together, we propose that SATB1 has functionally distinct modes of chromatin interaction by directly binding BURs to form a chromatin scaffold to which it indirectly tethers open chromatin. Such chromatin organization may provide a gene-regulatory network underlying cell type-specific gene expression.

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  1. eLife Assessment

    This important study has modified ChIP-seq and 4C-seq procedures with a urea step and shows that this drastically changes the pattern of chromatin interactions observed for SATB1 but not other proteins (CTCF, Jarid2, Suz12, Ezh2). Multiple controls make the data convincing. The findings shed new light on the role of SATB1 in genome organization and will be of interest to those who study chromosome structure and nuclear organization.

  2. Reviewer #1 (Public review):

    Summary:

    The nuclear protein SATB-1 was originally identified as a protein of the 'nuclear matrix', an aggregate of nuclear components that arose upon extracting nuclei with high salt. While the protein was assumed to have a global function in chromatin organization, it has subsequently been linked to a variety of pathological conditions, notably cancer. The mapping of the factor by conventional ChIP procedures showed strong enrichment in active, accessible chromatin, suggesting a direct role in gene regulation, perhaps in enhancer-promoter communication. These findings did not explain why SATB-1-chromatin interaction resisted the 2 M salt extraction during early biochemical fractionation of nuclei.

    The authors, who have studied SATB-1 for many years, now developed an unusual variation of the ChIP procedure, in which they purify crosslinked chromatin by centrifugation through 8 M urea. Remarkably, while they lose all previously mapped signals for SATB-1 in active chromatin, they now gain many binding events in silent regions of the genome, represented by lamin-associated domains (LADs).

    SATB-1 had previously been shown by the authors and others to bind to DNA with special properties, termed BUR (for 'base-unpairing regions'). BURs are AT-rich and apparently enriched in equally AT-rich LADs. The 'urea-ChIP' pattern is essentially complementary to the classical ChIP pattern. The authors now speculate that the previously known SATB-1 binding pattern, which does not overlap BURs particularly well, is due to indirect chromatin binding, whereas they consider the urea-ChIP profile that fits better to the BUR distribution on the chromosome to be due to direct binding.

    Building on the success with urea-ChIP the authors adapted the 4C-procedure of chromosome conformation mapping to work with urea-purified chromatin. The data suggest that BUR-bound SATB-1 in heterochromatin mediates long-distance interaction with loci in active chromatin. They close with a model, whereby SATB-1 tethers active chromatin to the nuclear lamina. Because cell type-specific differences are observed, they suggest that the SATB-1 interactions are functionally relevant.

    Strengths:

    Given the unusual finding of essentially mutually exclusive 'standard ChIP' and 'urea-ChIP' profiles for SATB-1, the authors conducted many appropriate controls. They showed that all SATB-1 peaks in urea-ChIP and 96% of peaks in standard-ChIP represent true signals, as they are not observed in a SATB-1 knockout cell line. They also show that urea-ChIP and standard ChIP yield similar profiles for CTCF. The data appear reproducible, judged by at least two replicates and triangulation. The SATB-1 KO cells provide a nice control for the specificity of signals, including those that arise from their elaborately modified 4C protocol.

    Weaknesses:

    The weaknesses mainly relate to missing qualifier statements and overinterpretations. I also found some aspects of the model not yet well supported by the data.

    (1) Under high urea conditions the BUR elements should be rendered single-stranded, and one wonders whether this has any effect on the procedure. The authors should alert the reader of these circumstances.

    (2) An important conclusion is that urea-ChIP reveals direct DNA binding events, whereas standard ChIP shows indirect binding (which is stripped off by urea). I do not yet see any evidence for direct binding. It cannot be excluded, for example, that the binding is RNA-mediated. The authors mention in passing that urea-ChIP material still contains (specific!) RNA. Given that this is a new procedure, the authors should document the RNA content of urea-ChIP and RNase-treat their samples prior to ChIP to monitor an RNA contribution.

    (3) An important aspect of the model is that SATB-1 tethers active genes to inactive LADs. However, in the 4C experiment the BUR elements used to anchor the looping are both in the accessible, active chromatin domain.

  3. Reviewer #2 (Public review):

    Summary:

    The report by Kohwi-Shigematsu et al. describes the key observation that SATB1 binds directly to so-called BUR elements. This is in contrast to several other reports describing SATB1 binding to promoters and enhancers. This discrepancy is explained by the authors to depend on the features of the ChIP technique being used. Urea-ChIP, innovated by the authors, strips off protein-protein interactions that are maintained in conventional ChIP. The authors convincingly make the case that SATB1 and the key genome organiser CTCF co-localize by conventional ChIP but not urea ChIP, as particularly evident in Figure 2A. SATB1 controls long-range interactions in thymocytes and the expression of gene clusters. This feature is independent of TADs, as the knockdown of SATB1 expression does not affect the TAD patterns.

    Strengths:

    A new and innovative adaptation of the urea ChIP-seq technique has enabled the authors to reveal a new aspect of SATB1 binding to the genome. The authors provide a wealth of data to reinforce their claims. This report thus sheds new light on SATB1 function, which is particularly important given its role in metastasising cancer cells.

    Weaknesses:

    No weaknesses were identified by this reviewer.